Novel piperidine compound or salt thereof

ABSTRACT

A piperidine compound represented by a general formula (I) or a salt thereof: wherein, R 1  represents a carboxyl group, —C(═O)NR 5 R 6 , or an oxadiazolyl group optionally having a C 1 -C 6  alkyl group or a trifluoromethyl group as a substituent; R 2  represents a halogen atom or a C 1 -C 6  alkoxy group; R 3  represents a phenyl group optionally having 1 to 3 same or different group(s) selected from a halogen atom, a C 1 -C 6  alkyl group, a C 1 -C 6  alkoxy group, and a trifluoromethyl group as a substituent; R 4  represents a hydrogen atom or a C 1 -C 6  alkyl group; and R 5  and R 6  are the same or different and each represent a hydrogen atom, a C 1 -C 6  alkyl group, or a C 3 -C 6  cycloalkyl group, or R 5  and R 6  optionally form a 3 to 6-membered nitrogen-containing saturated heterocyclic group together with a nitrogen atom to which R 5  and R 6  are bound, are provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a divisional of U.S. application Ser. No.15/131,372, filed Apr. 18, 2016, which is a divisional of U.S.application Ser. No. 14/381,814, filed Aug. 28, 2014, now issued as U.S.Pat. No. 9,346,787. The entire contents of the above applications areincorporated herein by reference. U.S. application Ser. No. 14/381,814is a 371 of International Application No. PCT/JP2013/055064, filed Feb.27, 2013 which is based upon and claims the benefits of priority toJapanese Application No. 2012-043303, filed Feb. 29, 2012 and JapaneseApplication No. 2012-186534, filed Aug. 27, 2012.

FIELD OF THE INVENTION

The present invention relates to a novel piperidine compound having theaurora A kinase inhibitory action or a salt thereof, and the use of thecompound or a salt thereof.

BACKGROUND OF THE INVENTION

Aurora A is a member of serine-threonine kinases, and is widely involvedin, for example, the formation and maturation of centrosomes, spindledynamics, and chromosome alignment in the mitotic phase (M phase) of thecell cycle, thereby regulating the progression of mitosis (Non PatentDocument 1). So far, overexpression and/or amplification of aurora Ahave been confirmed in a wide variety of carcinomas (Non Patent Document2). Also, since inhibition of aurora A kinase in tumor cells induces notonly termination of mitosis, but also apoptosis, aurora A is one of theimportant target molecules in cancer therapy.

Meanwhile, microtubule-targeting agents as represented by taxane andvinca alkaloid are widely used as the key drug in cancer chemotherapy.However, persistent and adequate therapeutic effects are not alwaysobtained due to loss of responsiveness to drugs or becoming resistanceto drugs. Therefore, there is clinical need for development of a drugcapable of potentiating the anti-tumor effect of taxane drugs since sucha drug promises to provide more effective therapeutic opportunities. Thecytocidal effect of taxane anticancer agents requires activation of thespindle assembly checkpoint in the cell cycle, and there is a reportthat tumor cells having a reduced spindle assembly checkpoint activityshow reduced sensitivity to taxane anticancer agents (Non PatentDocument 3). In addition, it is known that a cell line overexpressingaurora A becomes resistance to paclitaxel (Non Patent Document 4) andinhibition of aurora A potentiates the activity of paclitaxel ordocetaxel (Non Patent Document 5). Meanwhile, it has been reported thatalthough aurora B, which is a subtype thereof, shows activity on themitotic phase (M phase) of the cell cycle with aurora A, inhibition ofaurora B reduces the spindle assembly checkpoint activity (Non PatentDocument 6). Therefore, it is suggested that the inhibition of aurora Bmight attenuate the effect of taxane drugs. Also, aurora C is stronglyexpressed in, for example, testis or germ cells, and the results ofhuman genome analysis have shown that aurora C is important inSpermatogenesis (Non Patent Document 7). The aurora C is known tofunction as complementation to the function of aurora B in cell division(Non Patent Document 8). Similarly to inhibition of aurora B, inhibitionof aurora c induces aneuploidy in cells, leading to exhibiting phenotypewhich greatly differs from that exhibited by inhibition of aurora A, andpotentiation of the effect of taxane drugs cannot presumably beexpected. Furthermore, influence on the reproductive system cannot beoverlooked, and therefore, it is desirable that the drug does notexhibit the inhibitory activity on aurora C.

According to above, it is expected that by administering a drug whichselectively inhibits aurora A kinase in combination with a taxaneanticancer agent, the drug will effectively potentiate the anti-tumoractivity of the taxane anticancer agent, thereby enabling highertherapeutic effects.

Also, there is a report that the cell cycle termination activity inducedby paclitaxel is sustained for several days in a mouse tumor model intowhich a human cancer cell line is transplanted (Non Patent Document 9).Therefore, an agent for oral administration is considered to bedesirable when an aurora A inhibitor is to concomitantly administered,because of enabling continuous exposure.

So far, it has been reported that an aminopyridine derivative exhibitingthe inhibitory activity on aurora A can be orally administered (PatentDocument 1). However, although Patent Document 1 describes theinhibitory activity on aurora A and on cell proliferation in vitro, anydescriptions relating to the evaluation of oral administration of theabove compound are not found.

CITATION LIST Patent Document

-   [Patent Document 1] WO2009/104802

Non Patent Document

-   [Non Patent Document 1] Nat. Rev. Drug Discov., 8, pp. 547 to 566    (2009)-   [Non Patent Document 2] Cancer Treat. Rev., 34, pp. 175 to 182    (2008)-   [Non Patent Document 3] Mol. Cancer Ther., 5, pp. 2963 to 2969    (2006)-   [Non Patent Document 4] Cancer Cell, 3, pp. 51 to 62 (2003)-   [Non Patent Document 5] Cancer Res., 65, pp. 2899 to 2905 (2005)-   [Non Patent Document 6] Mol. Cancer Ther., 8, pp. 2046 to 2056    (2009)-   [Non Patent Document 7] Nature Genet. 39: pp. 661 to 665, 2007-   [Non Patent Document 8] Genes Cells 10: pp. 617 to 626, 2005-   [Non Patent Document 9] Cancer Res., 71, pp. 4608 to 4616 (2011)

SUMMARY OF THE INVENTION Problem to be Solved by the Invention

Accordingly, an object of the present invention is to provide a novelcompound which shows an excellent aurora A-selective inhibitory activityand is useful as an orally administrable anticancer agent. Further,another object of the present is to provide a novel agent forpotentiation of anti-tumor effect of microtubule-targeting agentscontaining a taxane anticancer agent, and a combination therapy.

Means for Solving the Problem

The present inventors conducted intensive research in order to solve theaforementioned objects. As a result, they found that piperidine compoundhaving a specific substituent on the pyridine ring shows an excellentaurora A-selective inhibitory activity and cancer cell proliferationinhibitory action, and is orally administrable. They further found thatsuch a piperidine compound remarkably potentiated the anti-tumor effectsof microtubule-targeting agents containing a taxane anticancer agent,thereby completing the present invention.

Specifically, the present invention provides a piperidine compoundrepresented by a general formula (I) or a salt thereof:

wherein, R₁ represents a carboxyl group, —C(═O)NR₅R₆, or an oxadiazolylgroup optionally having a C₁-C₆ alkyl group or a trifluoromethyl groupas a substituent;

R₂ represents a halogen atom or a C₁-C₆ alkoxy group;

R₃ represents a phenyl group optionally having 1 to 3 same or differentgroup(s) selected from a halogen atom, a C₁-C₆ alkyl group, a C₁-C₆alkoxy group, and a trifluoromethyl group as a substituent;

R₄ represents a hydrogen atom or a C₁-C₆ alkyl group; and

R₅ and R₆ are the same or different and each represent a hydrogen atom,a C₁-C₆ alkyl group, or a C₃-C₆ cycloalkyl group, or R₅ and R₆optionally form a 3 to 6-membered nitrogen-containing saturatedheterocyclic group together with a nitrogen atom to which R₅ and R₆ arebound.

The present invention also provides a drug comprising the piperidinecompound represented by the aforementioned general formula (I) or a saltthereof as an active ingredient.

The present invention also provides an aurora A-selective inhibitor, ananti-tumor agent, or an agent for potentiation of anti-tumor effect of amicrotubule-targeting agent, comprising the piperidine compoundrepresented by the aforementioned general formula (I) or a salt thereofas an active ingredient.

The present invention also provides use of the piperidine compoundrepresented by the aforementioned general formula (I) or a salt thereoffor the production of an aurora A-selective inhibitor, an anti-tumoragent, or an agent for potentiation of anti-tumor effect of amicrotubule-targeting agent.

The present invention also provides the piperidine compound representedby the aforementioned general formula (I) or a salt thereof forselective inhibition of aurora A, treatment of cancer, or potentiationof an anti-tumor effect of a microtubule-targeting agent.

The present invention also provides a method for selective inhibition ofaurora A, treatment of cancer, or potentiation of an anti-tumor effectof a microtubule-targeting agent, comprising administering an effectivedose of the piperidine compound represented by the aforementionedgeneral formula (I) or a salt thereof.

The present invention also provides a cancer therapeutic agentcomprising the piperidine compound represented by the aforementionedgeneral formula (I) or a salt thereof and a microtubule-targeting agent;a composition comprising the piperidine compound represented by theaforementioned general formula (I) or a salt thereof and amicrotubule-targeting agent for treatment of cancer; use of acomposition comprising the piperidine compound represented by theaforementioned general formula (I) or a salt thereof and amicrotubule-targeting agent for the production of a cancer treatmentdrug; and a method for treating cancer, comprising administering aneffective dose of the piperidine compound represented by theaforementioned general formula (I) or a salt thereof concomitantly withan effective dose of a microtubule-targeting agent.

The present invention further provides the aforementioned drug, auroraA-selective inhibitor, anti-tumor agent, or agent for potentiation ofanti-tumor effect of a microtubule agonist for oral administration; useof the aforementioned aurora A-selective inhibitor, anti-tumor agent, ormicrotubule-targeting agent for oral administration for the productionof an anti-tumor effect potentiator; the aforementioned compound or asalt thereof for the aforementioned aurora A-selective inhibition,treatment of cancer, or potentiation of an anti-tumor effect of amicrotubule-targeting agent by oral administration; and a method for theaforementioned aurora A-selective inhibition, treatment of cancer, orpotentiation of an anti-tumor effect of a microtubule-targeting agent,wherein administration is done by means of oral administration.

Effects of the Invention

The compound (I) of the present invention or a salt thereof shows anexcellent aurora A-selective inhibitory activity and cancer cellproliferation inhibitory activity, and is orally administrable. Thecompound (I) of the present invention or a salt thereof is useful notonly as an anti-tumor agent, but also for administration in combinationwith microtubule-targeting agents containing a taxane anticancer agent.

DETAILED DESCRIPTION OF THE INVENTION

In the compound (I) of the present invention, selection of R₂ isimportant in terms of the aurora A-selective inhibitory activity, oralabsorbability, anti-tumor activity by oral administration, andpotentiation of the anti-tumor effect of microtubule-targeting agentscontaining a taxane anticancer agent. The compound of the presentinvention is characterized in that R₂ is a halogen atom or a C₁-C₆alkoxy group.

In the specification of the present application, the “C₁-C₆ alkyl group”represents a linear or branched alkyl group having 1 to 6 carbon atoms,and specific examples thereof include a methyl group, an ethyl group, ann-propyl group, an isopropyl group, an n-butyl group, an isobutyl group,a sec-butyl group, a tert-butyl group, a pentyl group, and a hexylgroup. The C₁-C₆ alkyl group is preferably a linear or branched alkylgroup having 1 to 4 carbon atoms (C₁-C₄ alkyl group).

In the specification of the present application, the “oxadiazolyl group”refers to a 1,2,4-oxadiazolyl group or a 1,3,4-oxadiazolyl group. Theoxadiazolyl ring is preferably unsubstituted or substituted with a C₁-C₄alkyl group or a trifluoromethyl group, and the oxadiazolyl ring is morepreferably unsubstituted or substituted with a methyl group or atrifluoromethyl group.

In the specification of the present application, examples of the“halogen atom” include a fluorine atom, a chlorine atom, a bromine atom,and an iodine atom.

In the specification of the present application, the “C₁-C₆ alkoxygroup” represents a linear or branched alkoxy group having 1 to 6 carbonatoms, and specific examples thereof include a methoxy group, an ethoxygroup, an n-propoxy group, an isopropoxy group, an n-butoxy group, anisobutoxy group, a t-butoxy group, a pentoxy group, and a hexoxy group.

The C₁-C₆ alkoxy group is preferably a linear or branched alkoxy grouphaving 1 to 4 carbon atoms (C₁-C₄ alkoxy group).

In the specification of the present application, the “C₃-C₆ cycloalkylgroup” refers to a monocyclic cycloalkyl group having 3 to 6 carbonatoms, and specific examples thereof include a cyclopropyl group, acyclobutyl group, a cyclopentyl group, and a cyclohexyl group. The C₃-C₆cycloalkyl group is preferably a cyclopropyl group or a cyclobutylgroup.

In the specification of the present application, the phrase “R₅ and R₆optionally form a 3 to 6-membered nitrogen-containing saturatedheterocyclic group together with a nitrogen atom to which R₅ and R₆ arebound” means that R₅ and R₆ optionally form, together with a nitrogenatom to which R₅ and R₆ are bound (that is, as —NR₅R₆), a 3 to6-membered saturated heterocyclic group further containing 0 to 2nitrogen atom(s) and/or oxygen atom(s) within the ring. Specificexamples of the 3 to 6-membered nitrogen-containing saturatedheterocyclic group which is optionally formed include an azetidinylgroup, a pyrrolidinyl group, a piperidinyl group, a piperazinyl group, amorpholinyl group, and an isoxazolidinyl group.

In the general formula (I), R₁ is preferably a carboxyl group,—C(═O)NR₅R₆ (wherein, R₅ and R₆ are the same or different and eachrepresent a hydrogen atom, a C₁-C₆ alkyl group, or a C₃-C₆ cycloalkylgroup, or R₅ and R₆ optionally form an azetidinyl group, a pyrrolidinylgroup, or an isoxazolidinyl group together with a nitrogen atom to whichR₅ and R₆ are bound), or an oxadiazolyl group optionally having a C₁-C₆alkyl group or a trifluoromethyl group as a substituent.

In the general formula (I), R₁ is more preferably a carboxyl group,—C(═O)NR₅R₆ (wherein, R₅ and R₆ are the same or different and eachrepresent a hydrogen atom, a methyl group, a cyclopropyl group, or acyclobutyl group, or R₅ and R₆ represent an azetidinyl group, apyrrolidinyl group, or an isoxazolidinyl group together with a nitrogenatom to which R₅ and R₆ are bound), or an oxadiazolyl group optionallyhaving a methyl group or a trifluoromethyl group as a substituent.

As demonstrated in Examples later, it is important that R₂ in thegeneral formula (I) be a halogen atom or a C₁-C₆ alkoxy group in termsof the aurora A-selective inhibitory activity, oral absorbability,anti-tumor activity by oral administration, and a potentiation of theanti-tumor effect of microtubule-targeting agents including a taxaneanticancer agent. The R₂ is preferably a fluorine atom, a chlorine atom,or a C₁-C₄ alkoxy group, more preferably a fluorine atom, a chlorineatom, or a methoxy group.

In the general formula (I), R₃ is preferably a phenyl group optionallyhaving 1 to 3 same or different group(s) selected from a halogen atom, aC₁-C₄ alkyl group, a C₁-C₄ alkoxy group, and a trifluoromethyl group asa substituent, more preferably a phenyl group having 1 to 2 same ordifferent group(s) selected from the aforementioned substituents. R₃ iseven more preferably a phenyl group having 1 to 2 same or differentgroup(s) selected from a fluorine atom, a chlorine atom, a methyl group,a methoxy group, and a trifluoromethyl group as a substituent.

In the general formula (I), R₄ is preferably a hydrogen atom or a C₁-C₄alkyl group, and more preferably a hydrogen atom or a methyl group.

In the compound of the present invention, it is preferable that R₁ is acarboxyl group, —C(═O)NR₅R₆ (wherein, R₅ and R₆ are the same ordifferent and each represent a hydrogen atom, a C₁-C₄ alkyl group, or aC₃-C₆ cycloalkyl group, or R₅ and R₆ optionally form an azetidinylgroup, a pyrrolidinyl group, or an isoxazolidinyl group together with anitrogen atom to which R₅ and R₆ are bound), or an oxadiazolyl groupoptionally having a C₁-C₄ alkyl group or a trifluoromethyl group as asubstituent; R₂ is a fluorine atom, a chlorine atom, or a C₁-C₄ alkoxygroup; R₃ is a phenyl group optionally having 1 to 2 same or differentgroup(s) selected from a halogen atom, a C₁-C₄ alkyl group, a C₁-C₄alkoxy group, and a trifluoromethyl group as a substituent; and R₄ is ahydrogen atom or a C₁-C₄ alkyl group.

Also, as the compound of the present invention, a compound in which, inthe general formula (I), R₁ is a carboxyl group, —C(═O) NR₅R₆ (wherein,R₅ and R₆ are the same or different and each represent a hydrogen atom,a methyl group, a cyclopropyl group, or a cyclobutyl group, or R₅ and R₆represent an azetidinyl group, a pyrrolidinyl group, or anisoxazolidinyl group together with a nitrogen atom to which R₅ and R₆are bound), or an oxadiazolyl group optionally having a methyl group ora trifluoromethyl group as a substituent; R₂ is a fluorine atom, achlorine atom, or a methoxy group; R₃ is a phenyl group optionallyhaving 1 to 2 same or different group(s) selected from a fluorine atom,a chlorine atom, a methyl group, a methoxy group, and a trifluoromethylgroup as a substituent; and R₄ is a hydrogen atom or a methyl group ispreferable.

Also, a compound in which R₁ is a carboxyl group, —C(═O)NR₅R₆ (wherein,R₅ and R₆ are the same or different and each represent a hydrogen atomor a methyl group, or R₅ and R₆ represent an isoxazolidinyl grouptogether with a nitrogen atom to which R₅ and R₆ are bound), or a1,2,4-oxadiazolyl group or a 1,3,4-oxadiazolyl group optionally having amethyl group as a substituent; R₂ is a fluorine atom, a chlorine atom,or a methoxy group; R₃ is a phenyl group having 1 to 2 same or differentgroup(s) selected from a fluorine atom, a chlorine atom, a methyl group,a methoxy group, and a trifluoromethyl group as a substituent; and R₄ isa hydrogen atom or a methyl group is more preferable.

Further, a compound in which R₁ is a carboxyl group or a1,2,4-oxadiazolyl group optionally having a methyl group as asubstituent; R₂ is a fluorine atom; R₃ is a phenyl group having 1 to 2same or different group(s) selected from a fluorine atom and a chlorineatom as a substituent; and R₄ is a methyl group is particularlypreferable.

The following compounds can be given as examples of specific preferablecompounds of the present invention.

-   1-(2,3-dichlorobenzoyl)-4-((5-fluoro-6-(5-methyl-1H-pyrazol-3-ylamino)pyridin-2-yl)methyl)piperidine-4-carboxylic    acid (compound 1)-   1-(2-fluoro-3-trifluoromethylbenzoyl)-4-((5-fluoro-6-(5-methyl-1H-pyrazol-3-ylamino)pyridin-2-yl)methyl)piperidine-4-carboxylic    acid (compound 2)-   1-(3-chloro-2-fluorobenzoyl)-4-((5-fluoro-6-(1H-pyrazol-3-ylamino)pyridin-2-yl)methyl)piperidine-4-carboxylic    acid (compound 10)-   1-(3-chloro-2-fluorobenzoyl)-4-((5-methoxy-6-(5-methyl-1H-pyrazol-3-ylamino)pyridin-2-yl)methyl)piperidine-4-carboxylic    acid (compound 11)-   1-(3-chloro-2-fluorobenzoyl)-4-((5-chloro-6-(5-methyl-1H-pyrazol-3-ylamino)pyridin-2-yl)methyl)piperidine-4-carboxylic    acid (compound 12)-   1-(3-chloro-2-fluorobenzoyl)-4-((5-fluoro-6-(5-methyl-1H-pyrazol-3-ylamino)pyridin-2-yl)methyl)piperidine-4-carboxylic    acid (compound 13)-   1-(3-chloro-2-fluorobenzoyl)-4-((5-fluoro-6-(5-methyl-1H-pyrazol-3-ylamino)pyridin-2-yl)methyl)-N-methylpiperidine-4-carboxyamide    (compound 14)-   1-(3-chloro-2-fluorobenzoyl)-4-((5-fluoro-6-(5-methyl-1H-pyrazol-3-ylamino)pyridin-2-yl)methyl)-N,N-dimethylpiperidine-4-carboxyamide    (compound 16)-   azetidin-1-yl(1-(3-chloro-2-fluorobenzoyl)-4-((5-fluoro-6-(5-methyl-1H-pyrazol-3-ylamino)pyridin-2-yl)methyl)piperidin-4-yl)methanone    (compound 19)-   (1-(3-chloro-2-fluorobenzoyl)-4-((5-fluoro-6-(5-methyl-1H-pyrazol-3-ylamino)pyridin-2-yl)methyl)piperidin-4-yl)(isoxazolidin-2-yl)methanone    (compound 21)-   (3-chloro-2-fluorophenyl)(4-((5-fluoro-6-(5-methyl-1H-pyrazol-3-ylamino)pyridin-2-yl)methyl)-4-(5-methyl-1,2,4-oxadiazol-3-yl)piperidin-1-yl)methanone    (compound 22)-   (2,3-dichlorophenyl)(4-((5-fluoro-6-(5-methyl-1H-pyrazol-3-ylamino)pyridin-2-yl)methyl)-4-(5-methyl-1,2,4-oxadiazol-3-yl)piperidin-1-yl)methanone    (compound 23)-   (3-chloro-2-fluorophenyl)(4-((5-fluoro-6-(5-methyl-1H-pyrazol-3-ylamino)pyridin-2-yl)methyl)-4-(1,2,4-oxadiazol-3-yl)piperidin-1-yl)methanone    (compound 24)-   (3-chloro-2-fluorophenyl)(4-((5-fluoro-6-(5-methyl-1H-pyrazol-3-ylamino)pyridin-2-yl)methyl)-4-(5-methyl-1,3,4-oxadiazol-3-yl)piperidin-1-yl)methanone    (compound 28)-   (3-chloro-2-fluorophenyl)(4-((5-fluoro-6-(5-methyl-1H-pyrazol-3-ylamino)pyridin-2-yl)methyl)-4-(3-methyl-1,2,4-oxadiazol-5-yl)piperidin-1-yl)methanone    (compound 29)

Next, a representative production method of the compound (I) of thepresent invention is hereinafter illustrated.

The compound (I) of the present invention can be produced by, forexample, the following production method, a method shown in Examples.However, the production method of the compound (I) of the presentinvention is not limited to these reaction examples. The raw materialsnecessary for the synthesis of the compound of the present invention canbe obtained as commercial products or easily produced by a productionmethod described in, for example, prior art documents.

Among the compounds (I) of the present invention, a compound (I-1), inwhich R₁ is a carboxyl group, can be produced by, for example, thefollowing production method 1.

wherein, X and Y each represent a leaving group; P represents a hydrogenatom or a protecting group; and Z represents a general formula (a) or(b):

and R₂, R₃, and R₄ are defined as above.

In the above production method 1, examples of the leaving grouprepresented by X or Y include a halogen atom, and it is preferably abromine atom. Examples of the protecting group represented by P includea tert-butyl group, a methoxy methyl group, a[(2-trimethylsilyl)ethoxy]methyl group, and a benzyl group, and it ispreferably a tert-butyl group.

(Step 1)

This step is a method for producing a compound (IV) by reacting acompound (II) with a base, and then with a compound (III). Examples ofthe compound (III) to be used in this step include6-bromo-2-bromomethyl-5-fluoropyridine,6-bromo-2-chloromethyl-5-fluoropyridine,2-bromomethyl-6-chloro-5-fluoropyridine,2-bromomethyl-5,6-dichloropyridine, and6-bromo-2-bromomethyl-5-methoxypyridine, and it is preferably6-bromo-2-bromomethyl-5-fluoropyridine. The compound (III) can beobtained as a commercial product or produced in accordance with apublicly known method.

The amount of the compound (III) to be used in this step is 0.1 to 10equivalents, preferably 0.8 to 2 equivalents relative to one equivalentof compound (II). The reaction temperature is −90 to 100° C., preferably−78 to 0° C. The reaction time is 0.1 to 100 hours, preferably 0.5 to 10hours. Examples of the base include lithium diisopropylamine and lithiumhexamethyldisilazide, and the base can be used in an amount of 0.5 to 10equivalents, preferably 1 to 1.5 equivalents. The solvent to be used inthis reaction is not particularly limited as long as it does notinterfere with the reaction, and examples thereof includetetrahydrofuran, 2-methyltetrahydrofuran, diethyl ether,1,2-dimethoxyethane, and toluene. These solvents can be used alone or asa mixture thereof.

The compound (IV) to be obtained as above can be subjected to thesubsequent step with or without isolation and purification by a publiclyknown isolation and purification methods such as concentration,concentration under reduced pressure, crystallization, solventextraction, reprecipitation, and chromatography.

(Step 2)

This step is a method for producing a compound (VI) by a couplingreaction between the compound (IV) and a compound (V). Examples of thecompound (V) to be used in this step (compound (V-1) or compound (V-2))include 1-tert-butyl-3-methyl-1H-pyrazol-5-amine,1-tert-butyl-1H-pyrazol-5-amine,1-{[2-(trimethylsilyl)ethoxy]methyl}-1H-pyrazol-3-amine, and5-methyl-1-{[2-(trimethylsilyl)ethoxy]methyl}-1H-pyrazol-3-amine. Thecompound (V) can be obtained as a commercial product or produced inaccordance with a publicly known method.

The amount of the compound (V) to be used in this step is 0.5 to 10equivalents, preferably 0.8 to 2 equivalents relative to one equivalentof compound (IV). Examples of a catalyst to be used include a metalcatalyst such as tris(benzylideneacetone)dipalladium and palladiumacetate, and the catalyst can be used in an amount of 0.001 to 5equivalents, preferably 0.005 to 0.1 equivalent relative to oneequivalent of compound (IV). Examples of a ligand for the aforementionedmetal catalyst include 4,5-bis(diphenylphosphino)-9,9-dimethylxantheneand 2,2′-bisdiphenylphosphino-1,1′-binaphthyl, and these ligands can beused in an amount of 0.001 to 5 equivalents, preferably 0.005 to 0.2equivalent relative to one equivalent of compound (IV). The reactiontemperature is 0 to 200° C., preferably room temperature to 130° C. Thereaction time is 0.1 to 100 hours, preferably 0.5 to 20 hours. Examplesof the base include an inorganic base such as potassium phosphate,sodium carbonate, potassium carbonate, cesium carbonate, and sodiumtert-butoxide, and organic amines such as trimethylamine,diisopropylethylamine, and pyridine, and these bases can be used in anamount of 0.5 to 10 equivalents, preferably 1 to 3 equivalents. Thesolvent to be used in this reaction is not particularly limited as longas it does not interfere with the reaction, and examples thereof includetoluene, tetrahydrofuran, dioxane, N,N-dimethylformamide,N,N-dimethylacetamide, N-methylpyrrolidin-2-one, tert-butanol, andtert-amyl alcohol. These solvents can be used alone or as a mixturethereof.

The compound (VI) to be obtained as above can be subjected to thesubsequent step with or without isolation and purification by a publiclyknown isolation and purification methods such as concentration,concentration under reduced pressure, crystallization, solventextraction, reprecipitation, and chromatography.

(Step 3)

This step is a method for producing a compound (VII) by removing thetert-butoxycarbonyl group, which is the protecting group for thecompound (VI), in the presence of an acid. With regard to the reactionconditions used in this step, this step can be carried out in accordancewith the method described in the Document (Protective Groups in OrganicSynthesis, written by T. W. Greene, John Wiley & Sons, Inc. (1981)) or amethod equivalent to the above method. Examples of the acid to be usedinclude trifluoroacetic acid, hydrochloric acid, sulfuric acid,methanesulfonic acid, and toluenesulfonic acid, and the acid can be usedin an amount of 0.1 to 100 equivalents, preferably 1 to 10 equivalents.The reaction temperature is 0 to 200° C., preferably room temperature to100° C. The reaction time is 0.1 to 100 hours, preferably 0.5 to 20hours. The solvent to be used in this reaction is not particularlylimited as long as it does not interfere with the reaction, and examplesthereof include chloroform, acetonitrile, toluene, tetrahydrofuran,dioxane, water, and acetic acid. These solvents can be used alone or asa mixture thereof.

The compound (VII) to be obtained as above can be subjected to thesubsequent step with or without isolation and purification by a publiclyknown isolation and purification methods such as concentration,concentration under reduced pressure, crystallization, solventextraction, reprecipitation, and chromatography.

(Step 4)

This step is a reaction for obtaining a compound (IX) by a dehydrationcondensation reaction between the compound (VII) and a compound (VIII).Examples of the compound (VIII) to be used in this step include2-fluoro-3-chlorobenzoic acid and 2,3-dichlorobenzoic acid. The compound(VIII) can be obtained as a commercial product or produced in accordancewith a publicly known method. In this step, using a commonly usedcondensing agent, the compound (IX) can be obtained in accordance with apublicly known method. Examples of the condensing agent includeN,N′-dicyclohexylcarbodiimide (DCC), N,N′-diisopropylcarbodiimide (DIC),1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (WSC),diphenylphosphoryl azide (DPPA),(benzotriazol-1-yl-oxy)trisdimethylaminophosphonium hexafluorophosphate(BOP), (benzotriazol-1-yl-oxy)tripyrrolidinophosphoniumhexafluorophosphate (PyBOP),(7-azabenzotriazol-1-yloxy)trispyrrolidinophosphonium phosphate (PyAOP),bromotrispyrrolidinophosphonium hexafluorophosphate (BroP),chlorotris(pyrrolidin-1-yl)phosphonium hexafluorophosphate (PyCroP),3-(diethoxyphosphoryloxy)-1,2,3-benzotriazin-4(3H)-one (DEPBT),0-(benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate(HATU), and 4-(5,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinehydrochloride (DMTMM). Examples of an additive to be used in this stepinclude 1-hydroxybenzotriazol (HOBt), 1-hydroxy-7-azabenzotriazole(HOAt), and N-hydroxysuccinimide (HOSu). These additives can be used inan amount of 0.1 to 100 equivalents, preferably 1 to 10 equivalents. Ifnecessary, a base such as trimethylamine, triethylamine, tripropylamine,diisopropylethylamine, N-methylmorpholine, pyridine,4-(N,N-dimethylamino)pyridine, lutidine, and collidine can be used in anamount of 0.1 to 100 equivalents, preferably 1 to 10 equivalents. Thesolvent is not particularly limited, and for example, water, methanol,ethanol, 2-propanol, tetrahydrofuran, 1,4-dioxane, toluene, methylenechloride, chloroform, acetonitrile, N,N-dimethylformamide, andN,N-dimethylacetamide, dimethyl sulfoxide can be used. The reactiontemperature is −30 to 200° C., preferably 0 to 50° C. The reaction timeis 0.1 to 100 hours, preferably 0.5 to 24 hours.

The compound (IX) to be obtained as above can be subjected to thesubsequent step with or without isolation and purification by a publiclyknown isolation and purification methods such as concentration,concentration under reduced pressure, crystallization, solventextraction, reprecipitation, and chromatography.

(Step 5)

This step is a method for producing a compound (I-1) by simultaneouslycarrying out hydrolysis of the cyano group of the compound (IX) andremoval of the protecting group (P) of the substituent Z under theacidic conditions. Examples of an acid to be used include hydrochloricacid, sulfuric acid, methanesulfonic acid, toluenesulfonic acid, andtrifluoroacetic acid. These acids can be used in an amount of 0.1 to 100equivalents, preferably 1 to 10 equivalents. The reaction temperature isroom temperature to 200° C., preferably 60 to 130° C. The reaction timeis 0.1 to 100 hours, preferably 0.5 to 20 hours. The solvent to be usedin this reaction is not particularly limited as long as it does notinterfere with the reaction, and examples thereof include dioxane,water, acetic acid, toluene, tetrahydrofuran, and 2-propanol. Thesesolvents can be used alone or as a mixture thereof. The compound (I-1)to be obtained as above can be isolated and purified by a publicly knownisolation and purification methods such as concentration, concentrationunder reduced pressure, crystallization, solvent extraction,reprecipitation, and chromatography.

Among the compounds of the general formula (I), a compound (I-2), inwhich R₁ is —C(═O)NR₅R₆, can be produced by, for example, the followingproduction method 2.

wherein, R₂, R₃, R₄, R₅, and R₆ are defined as above.

(Step 6)

This step is a method for producing the compound (I-2) by a dehydrationcondensation reaction between the compound (I-1) obtained by theproduction method 1 and a compound (X). Examples of the compound (X) tobe used in this step include an amine such as methylamine,dimethylamine, ammonium chloride, cyclopropylamine, and pyrrolidine aswell as salts thereof. The compound (X) can be obtained as a commercialproduct or produced in accordance with a publicly known method. In thisstep, using a commonly used condensing agent, the compound (I-2) can beobtained in accordance with a publicly known method. Examples of thecondensing agent include N,N′-dicyclohexylcarbodiimide (DCC),N,N′-diisopropylcarbodiimide (DIC),1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (WSC),diphenylphosphoryl azide (DPPA),(benzotriazol-1-yl-oxy)trisdimethylaminophosphonium hexafluorophosphate(BOP), (benzotriazol-1-yl-oxy)tripyrrolidinophosphoniumhexafluorophosphate (PyBOP),(7-azabenzotriazol-1-yloxy)trispyrrolidinophosphonium phosphate (PyAOP),bromotrispyrrolidinophosphonium hexafluorophosphate (BroP),chlorotris(pyrrolidin-1-yl)phosphonium hexafluorophosphate (PyCroP),3-(diethoxyphosphoryloxy)-1,2,3-benzotriazin-4(3H)-one (DEPBT),O-(benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate(HATU), and 4-(5,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinehydrochloride (DMTMM). Examples of an additive to be used in this stepinclude 1-hydroxybenzotriazol (HOBt), 1-hydroxy-7-azabenzotriazole(HOAt), and N-hydroxysuccinimide (HOSu). These additives can be used inan amount of 0.1 to 100 equivalents, preferably 1 to 10 equivalents. Ifnecessary, a base such as trimethylamine, triethylamine, tripropylamine,diisopropylethylamine, N-methylmorpholine, pyridine,4-(N,N-dimethylamino)pyridine, lutidine, and collidine can be used in anamount of 0.1 to 100 equivalents, preferably 1 to 10 equivalents. Thesolvent is not particularly limited, and for example, water, methanol,ethanol, 2-propanol, tetrahydrofuran, 1,4-dioxane, toluene, methylenechloride, chloroform, acetonitrile, N,N-dimethylformamide,N,N-dimethylacetamide, and dimethyl sulfoxide can be used. The reactiontemperature is −30 to 200° C., preferably 0 to 50° C. The reaction timeis 0.1 to 100 hours, preferably 0.5 to 24 hours.

The compound (I-2) to be obtained as above can be isolated and purifiedby a publicly known isolation and purification methods such asconcentration, concentration under reduced pressure, crystallization,solvent extraction, reprecipitation, and chromatography.

Among the compounds of the general formula (I), a compound (I-3), inwhich R₁ is 1,2,4-oxadiazol substituted with R₇, can be produced by, forexample, the following production method 3.

wherein, R₇ is a C₁-C₆ alkyl group or a trifluoromethyl group; R₂, R₃,R₄, R₅, R₆, and Z are defined as above.

(Step 7)

This step is a method for producing a compound (XI) by converting thecyano group in the compound (VI), which is obtained as a productionintermediate in the production method 1, into amidoxime. The reactionsused in this step can be carried out by, for example, the methodsdescribed in International Publication No. WO2005/026123, InternationalPublication No. WO2008/117175, International Publication No.WO2008/156721, or in accordance with a method equivalent to thosemethods. For example, the reactions can be carried out by reacting thecompound (VI) with hydroxylamine in an alcoholic solvent such as ethanoland 2-propanol. If necessary, a base can be used, and examples of thebase include organic amines such as triethylamine,diisopropylethylamine, N-methylmorpholine, and pyridine and inorganicsalts such as sodium bicarbonate, sodium carbonate, potassium carbonate,cesium carbonate, sodium methoxide, sodium ethoxide, and potassiumtert-butoxide. Hydroxylamine can be used in an amount of 1 to 100equivalents, preferably 1 to 10 equivalents. The reaction temperature isroom temperature to 150° C., preferably 50 to 100° C. The reaction timeis 0.1 to 100 hours, preferably 0.5 to 20 hours.

The compound (XI) to be obtained as above can be subjected to thesubsequent step with or without isolation and purification by a publiclyknown isolation and purification methods such as concentration,concentration under reduced pressure, crystallization, solventextraction, reprecipitation, and chromatography.

(Step 8)

This step is a method for producing a compound (XII) by converting theamidoxime group in the compound (XI) into a 1,2,4-oxadiazole ring. Thereactions used in this step can be carried out by, for example, themethods described in International Publication No. WO2005/026123,International Publication No. WO2008/117175, International PublicationNo. WO2008/156721, or in accordance with a method equivalent to thosemethods. For example, the reactions can be carried out by reacting thecompound (XI) with acetic anhydride, acetyl chloride, triethylorthoformate, and triethyl orthoacetate in a solvent such as toluene,chloroform, acetic acid, N,N-dimethylformamide,N-methylpyrrolidin-2-one, and pyridine. If necessary, a base can beused, and examples of the base include triethylamine,diisopropylethylamine, N-methylmorpholine, and pyridine. The reactiontemperature is room temperature to 150° C., preferably 50 to 100° C. Thereaction time is 0.1 to 100 hours, preferably 0.5 to 20 hours.

The compound (XII) to be obtained as above can be subjected to thesubsequent step with or without isolation and purification by a publiclyknown isolation and purification methods such as concentration,concentration under reduced pressure, crystallization, solventextraction, reprecipitation, and chromatography.

(Step 9)

This step is a method for producing a compound (XIII) by removing thetert-butoxycarbonyl group, which is the protecting group for thecompound (XII), in the presence of an acid. This step can be carried outby a method similar to that used in the aforementioned step 3 or amethod equivalent to that used in the step 3.

The compound (XIII) to be obtained as above can be subjected to thesubsequent step with or without isolation and purification by a publiclyknown isolation and purification methods such as concentration,concentration under reduced pressure, crystallization, solventextraction, reprecipitation, and chromatography.

(Step 10)

This step is a method for producing a compound (XIV) by a dehydrationcondensation reaction between the compound (XIII) and the compound(VIII). This step can be carried out by a method similar to that used inthe aforementioned step 4 or a method equivalent to that used in thestep 4.

The compound (XIV) to be obtained as above can be subjected to thesubsequent step with or without isolation and purification by a publiclyknown isolation and purification methods such as concentration,concentration under reduced pressure, crystallization, solventextraction, reprecipitation, and chromatography.

(Step 11)

This step is a method for producing a compound (I-3) by removing theprotecting group (P) of the substituent Z in the compound (XIV) in thepresence of an acid. This step can be carried out by a method similar tothat used in the aforementioned step 5 or a method equivalent to thatused in the step 5.

The compound (I-3) to be obtained as above can be isolated and purifiedby a publicly known isolation and purification methods such asconcentration, concentration under reduced pressure, crystallization,solvent extraction, reprecipitation, and chromatography.

Among the compounds of the general formula (I), a compound (I-4), inwhich R₁ is 1,3,4-oxadiazol substituted with R₇, can be produced by, forexample, the following production method 4.

wherein, R₇ represents a C₁-C₆ alkyl group or a trifluoromethyl group;R₂, R₃, and R₄ are defined as above.

(Step 12)

This step is a method for producing a compound (XV) by a dehydrationcondensation reaction between the compound (I-1) obtained by theproduction method 1 and tert-butoxycarbonyl hydrazide. This step can becarried out by a method similar to that used in the aforementioned step4 or a method equivalent to that used in the step 4.

The compound (XV) to be obtained as above can be subjected to thesubsequent step with or without isolation and purification by a publiclyknown isolation and purification methods such as concentration,concentration under reduced pressure, crystallization, solventextraction, reprecipitation, and chromatography.

(Step 13)

This step is a method for producing a compound (XVI) by removing thetert-butoxycarbonyl group, which is the protecting group for thecompound (XV), in the presence of an acid. This step can be carried outby a method similar to that used in the aforementioned step 3 or amethod equivalent to that used in the step 3.

The compound (XVI) to be obtained as above can be subjected to thesubsequent step with or without isolation and purification by a publiclyknown isolation and purification methods such as concentration,concentration under reduced pressure, crystallization, solventextraction, reprecipitation, and chromatography.

(Step 14)

This step is a method for producing a compound (I-4) by converting theacyl hydrazide group in the compound (XVI) into a 1,3,4-oxadiazole ring.This step can be carried out by a method similar to that used in theaforementioned step 8 or a method equivalent to that used in the step 8.

The compound (I-4) to be obtained as above can be subjected to thesubsequent step with or without isolation and purification by a publiclyknown isolation and purification methods such as concentration,concentration under reduced pressure, crystallization, solventextraction, reprecipitation, and chromatography.

When the compound of the present invention includes isomers such asoptical isomers, stereoisomers, position isomers, and rotationalisomers, a mixture of any isomer is also encompassed by the compound ofthe present invention. For example, when optical isomers exist for thecompound of the present invention, the optical isomers which areseparated from racemic forms are also encompassed by the compound of thepresent invention. These isomers can be individually obtained as asingle compound by a synthetic technique or separation technique knownper se (such as concentration, solvent extraction, columnchromatography, and recrystallization).

The compound of the present invention or a salt thereof may be acrystal, and a single crystal form as well as a polymorphic mixture isencompassed by the compound of the present invention or a salt thereof.A crystal can be produced by carrying out crystallization by applying acrystallization method known per se. The compound of the presentinvention or a salt thereof may be a solvate (such as a hydrate) or anon-solvate, and both of which are encompassed by the compound of thepresent invention or a salt thereof. A compound labeled with, forexample, an isotope (for example, ²H, ³H, ¹³C, ¹⁴C, ¹⁸F, ³⁵S, and ¹²⁵I)is also encompassed by the compound of the present invention or a saltthereof.

The salt of the compound of the present invention refers to a commonsalt used in the field of organic chemistry, and examples thereofinclude salts such as, when a compound has a carboxyl group, a baseaddition salt of the carboxyl group, and when a compound has an aminogroup or a basic heterocyclic group, an acid addition salt of the aminogroup or the basic heterocyclic group.

Examples of the base addition salt include an alkali metal salt such asa sodium salt and a potassium salt; an alkaline earth metal salt such asa calcium salt and a magnesium salt; an ammonium salt; and an organicamine salt such as a trimethylamine salt, a triethylamine salt, adicyclohexylamine salt, an ethanolamine salt, a diethanolamine salt, atriethanolamine salt, a procaine salt, and anN,N′-dibenzylethylenediamine salt.

Examples of the acid addition salt include an inorganic acid salt suchas a hydrochloride, a sulfate, a nitrate, a phosphate, and aperchlorate; an organic acid salt such as an acetate, a formate, amaleate, a fumarate, a tartrate, a citrate, an ascorbate, and atrifluoroacetate; and a sulfonate such as a methanesulfonate, anisethionate, a benzenesulfonate, and a p-toluenesulfonate.

The compound of the present invention or a salt thereof shows anexcellent selective aurora A inhibitory activity, and in particular,shows an extremely stronger selective aurora A inhibitory activitycompared to the inhibitory activity on aurora B and aurora C, and thusis useful as an aurora A-selective inhibitor. Further, the compound ofthe present invention or a salt thereof shows an excellent anti-tumoreffect, and thus is useful as an anti-tumor agent. Although cancer to betreated is not particularly limited, examples thereof include head andneck cancer, esophageal cancer, stomach cancer, duodenal cancer, coloncancer, rectal cancer, liver cancer, gallbladder and bile duct cancer,biliary tract cancer, pancreatic cancer, lung cancer, breast cancer,ovary cancer, uterine cervical cancer, uterine cancer, renal cancer,bladder cancer, prostate cancer, testicular cancer, bone and soft tissuesarcoma, hematologic cancer, multiple myeloma, skin cancer, brain tumor,mesothelioma, and hematologic cancer. Preferably, cancer to be treatedis hematologic cancer such as B cell lymphoma, chronic lymphocyticleukemia, peripheral T-cell lymphoma, myelodysplastic syndrome, acutemyelogenous leukemia, acute lymphocytic leukemia, and multiple myeloma,stomach cancer, breast cancer, prostate cancer, ovary cancer, lungcancer, and colon cancer. Additionally, the drug of the presentinvention can be applied to humans and animals other than humans.

Also, given that the compound of the present invention or a salt thereofhas an excellent aurora A-selective inhibitory activity, when it is usedin combination with a microtubule-targeting agent, it potentiates theanti-tumor effect of the microtubule-targeting agent. Therefore, thecompound of the present invention or a salt thereof is useful as ananti-tumor effect potentiator for a microtubule-targeting agent. Acomposition containing the compound of the present invention or a saltthereof and a microtubule agonist is useful as an anti-tumor agent(cancer treatment drug). Combinational administration of the compound ofthe present invention or a salt thereof and a microtubule-targetingagent is useful as a method for treating cancer. Examples of themicrotubule-targeting agent include a microtubule stabilizing drug suchas a taxane anticancer agent and an epothilone anticancer agent, andpreferably, the microtubule-targeting agent is a taxane anticanceragent. Examples of the taxane anticancer agent include paclitaxel,docetaxel, and cabazitaxel, and preferably, the taxane anticancer agentis paclitaxel. Examples of the epothilone anticancer agent includeepothilone B and epothilone D. The anti-tumor effect potentiator of thepresent invention can be administered at any time, i.e., before, orafter, or simultaneously with the administration of themicrotubule-targeting agent. Preferably, the anti-tumor effectpotentiator of the present invention may be administered at the sametime with, or within four hours before or after administration of themicrotubule-targeting agent. When the anti-tumor effect potentiator ofthe present invention is administered separately from or simultaneouslywith the microtubule agonist, for example, the anti-tumor effectpotentiator may be administered in such an amount that the amount of atleast one component selected from the compounds of the present inventionor salts thereof is in a range of 0.01 to 100 moles, preferably 0.05 to50 moles, more preferably 0.1 to 20 moles relative to one mole of themicrotubule-targeting agent. Although cancer to be treated is notparticularly limited, examples thereof include head and neck cancer,esophageal cancer, stomach cancer, duodenal cancer, colon cancer, rectalcancer, liver cancer, gallbladder and bile duct cancer, biliary tractcancer, pancreatic cancer, lung cancer, breast cancer, ovary cancer,uterine cervical cancer, uterine cancer, renal cancer, bladder cancer,prostate cancer, testicular cancer, bone and soft tissue sarcoma,hematologic cancer, multiple myeloma, skin cancer, brain tumor,mesothelioma, and hematologic cancer. Preferably, cancer to be treatedis hematologic cancer such as B cell lymphoma, chronic lymphocyticleukemia, peripheral T-cell lymphoma, myelodysplastic syndrome, acutemyelogenous leukemia, acute lymphocytic leukemia, and multiple myeloma,uterine cervical cancer, stomach cancer, breast cancer, prostate cancer,ovary cancer, lung cancer, and colon cancer. Additionally, the drug ofthe present invention can be applied to humans and animals other thanhumans.

Also, according to the present invention, it is possible to combine acomponent selected from the group consisting of the compounds of thepresent invention or salts thereof, which are the active ingredient ofthe aforementioned anti-tumor effect potentiator, with amicrotubule-targeting agent to prepare an anticancer agent formulatedwith an anti-tumor effect potentiator. In this case, the anticanceragent can be applied in the form of a mixed formulation containing thetotal active ingredient consisting of the microtubule-targeting agentand a component selected from the group consisting of the compounds ofthe present invention or salts thereof in a single preparation,alternatively, the anticancer agent can be prepared in the form of aseparate preparation each individually containing these activeingredients, alternatively, the anticancer agent can be prepared as akit formulation.

When the compound of the present invention or a salt thereof is used asa drug, a pharmaceutical carrier can be blended as needed to prepare apharmaceutical composition. Various dosage forms can be adoptedaccording to purpose of prevention or treatment. As the dosage form, forexample, any of an oral agent, an injection, a suppository, an ointment,and a patch is possible. The compound of the present invention or a saltthereof has excellent oral absorbability and exhibits an excellentanti-tumor activity through oral administration. In light of the above,an oral agent is preferably adopted. These dosage forms can be eachproduced by drug preparation methods which are publicly known andcommonly used by those skilled in the art.

As the pharmaceutical carrier, various kinds of organic or inorganiccarrier substances commonly used as pharmaceutical materials are used,and the pharmaceutical carrier is blended in a solid preparation as forexample an excipient, a binder, a disintegrant, a lubricant, and acolorant, or in a liquid preparation as for example a solvent, asolubilizing aid, a suspending agent, an isotonizing agent, a buffer,and a soothing agent. Also, an additive for drug preparation such as apreservative, an antioxidant, a colorant, a sweetener, and a stabilizingagent can also be used as needed.

When an oral solid preparation is prepared, an excipient, and ifnecessary, for example, an excipient, a binder, a disintegrant, alubricant, a colorant, and a corrigent are added to the compound of thepresent invention, and then, for example, a tablet, a coated tablet, agranule, a powder, and a capsule can be produced by a routine procedure.When an injection is prepared, for example, a pH adjusting agent, abuffer, a stabilizer, an isotonizing agent, and a local anesthetic areadded to the compound of the present invention, and a subcutaneous,intramuscular, and intravenous injection can be produced by a routineprocedure.

The amount of the compound of the present invention to be incorporatedin each of the aforementioned dosage unit forms is not constant butdepends on, for example, the symptoms of the patient to whom thecompound is applied, and on the dosage form of the compound. However,generally, the amount per dosage unit form is desirably 0.05 to 1000 mgfor an oral agent, desirably 0.01 to 500 mg for an injection, anddesirably 1 to 1000 mg for a suppository.

Also, the daily dose of a drug having the aforementioned dosage formvaries depending on, for example, the symptoms, body weight, age, or sexof a patient, and thus cannot be generally determined. However, thedaily dose of the compound of the present invention for a normal adult(weighing 50 kg) may be 0.05 to 5000 mg, preferably 0.1 to 1000 mg, andthe drug is preferably administered once a day or approximately twice orthree times a day in divided doses.

EXAMPLES

Hereinafter, the present invention is specifically described by Examplesand Test Examples. However, the present invention is not limited tothese Examples.

For various reagents used in Examples, commercial products were usedunless otherwise noted. For silica gel column chromatography,Purif-Pack® SI manufactured by Schott Moritex Corporation, KP-Sil®silica prepacked column manufactured by Biotage, or HP-Sil® silicaprepacked column manufactured by Biotage was used. For basic silica gelcolumn chromatography, Purif-Pack® NH manufactured by Schott MoritexCorporation or KP-NH® prepacked column manufactured by Biotage was used.For preparative thin-layer chromatography, Kieselgel™ 60F254, Art. 5744manufactured by Merck or NH2 silica gel 60F254 plate manufactured byWako Pure Chemical Industries, Ltd. was used. NMR spectra were measuredwith AL400 (400 MHz; JEOL, Ltd.), the Mercury 400 (400 MHz; AgilentTechnologies, Inc.) type spectrometer, or the Inova 400 (400 MHz;Agilent Technologies, Inc.) type spectrometer equipped with the OMNMRprobe (Protasis) by using, as the internal standard, tetramethylsilanewhen it was contained in a deuterated solvent, or by using, as theinternal standard, a NMR solvent in any other case, and all the δ valueswere expressed as ppm. The microwave reactions were carried out usingInitiator 8 manufactured by Biotage.

Also, for LCMS spectra, ACQUITY SQD (quadrupole) manufactured by WatersCorporation was used.

The abbreviations have the following meaning.

s: Singlet d: Doublet t: Triplet

dd: Double doublet

m: Multiplet br: Broad

brs: Broad singletDMSO-d₆: Deuterated dimethyl sulfoxideCDCl₃: Deuterated chloroformCD₃OD: Deuterated methanolXantphos: 4,5-Bis(diphenylphosphino)-9,9-dimethylxanthenePd₂(dba)₃: Tris(dibenzylideneacetone)dipalladium (0)K₃PO₄: Tripotassium phosphateMsOH: Mesylic acid

AIBN: Azobisisobutyronitrile HPMC: Hydroxypropylmethylcellulose Example1 Synthesis of1-(2,3-dichlorobenzoyl)-4-((5-fluoro-6-(5-methyl-1H-pyrazol-3-ylamino)pyridin-2-yl)methyl)piperidine-4-carboxylicAcid (Compound 1) (Step a) Synthesis of tert-butyl4-((6-bromo-5-fluoropyridin-2-yl)methyl)-4-cyanopiperidine-1-carboxylate

N-Boc-4-cyanopiperidine (5.35 g, 25.4 mmol) was dissolved in 100 mL oftetrahydrofuran. After cooling the resulting mixture to −78° C., asolution of lithium diisopropylamide/tetrahydrofuran complex incyclohexane (1.5 M, 16.5 mL, 24.8 mmol) was added while keeping theinternal temperature at or below −70° C. The resulting reaction mixturewas stirred at −78° C. for 20 minutes. To the reaction mixture thusobtained, 10 mL of a solution of2-bromo-6-(bromomethyl)-3-fluoropyridine in THF (6.28 g, 23.4 mmol) wasadded while keeping the internal temperature at or below −70° C.,followed by stirring at −78° C. for 20 minutes. To this reactionsolution, a mixture of hydrochloric acid (5M, 4.95 mL, 24.8 mmol) and 95mL of a saturated aqueous solution of ammonium chloride was added,followed by stirring at room temperature and then extraction with ethylacetate. The extract thus obtained was washed with saturated brine anddried over anhydrous sodium sulfate, and then filtered and concentrated.The tarry residue was dissolved in 6 mL of ethyl acetate, and 50 mL ofheptane was added dropwise while stirring. Seed crystals were thenadded, followed by stirring at room temperature for one hour. To theresulting light yellow suspension, 50 mL of heptane was further addeddropwise, followed by stirring overnight. The solid thus obtained wascollected by filtration and washed with a solution of ethyl acetate inheptane, and then dried under reduced pressure, whereby the titledproduct was obtained as an off-white solid (7.10 g, 17.8 mmol) (yield76%). The physical property values are shown below.

¹H-NMR (CDCl₃) δ: 7.45 (1H, t, J=8.1 Hz), 7.31 (1H, dd, J=8.1, 3.5 Hz),4.16 (2H, br), 3.09-2.93 (2H, m), 3.04 (2H, s), 1.95-1.84 (2H, m),1.68-1.57 (2H, m), 1.48 (9H, s); ESI-MS m/z 298, 300 (MH+).

(Step b) Synthesis of tert-butyl4-((6-(1-tert-butyl-3-methyl-1H-pyrazol-5-ylamino)-5-fluoropyridin-2-yl)methyl)-4-cyanopiperidine-1-carboxylate

The compound (6.37 g, 16.0 mmol) obtained by the aforementioned step a,5-amino-1-t-butyl-3-methylpyrazole (2.42 g, 15.8 mmol), xantphos (65.9mg, 114 μmol), Pd₂(dba)₃ (51.1 mg, 55.8 μmol), and K₃PO₄ (3.63 g, 17.1mmol) were placed in a reaction container, and 50 mL of toluene wasadded at last, followed by deaeration and argon substitution. Themixture thus obtained was stirred at 110° C. for eight hours, followedby addition of 200 mL of ethyl acetate at room temperature. The mixturethus obtained was washed with water and saturated brine and dried oversodium sulfate, and then filtered and concentrated. The residue wasdissolved in 10 mL of ethyl acetate, and 40 mL of heptane was addedwhile stirring at 75° C., followed by stirring at room temperatureovernight. The resulting solid was collected by filtration and washedwith 15% ethyl acetate/heptane, and then dried under reduced pressure,whereby the titled compound (4.15 g, 8.81 mmol) was obtained as a whitesolid (yield 56%). The physical property values are shown below.

¹H-NMR (CDCl₃) δ: 7.26 (1H, dd, J=10.7, 8.0 Hz), 6.74 (1H, dd, J=8.0,3.2 Hz), 6.23-6.15 (2H, m), 4.19-3.92 (2H, m), 3.09-2.92 (2H, m), 2.85(2H, s), 2.26 (3H, s), 1.95-1.86 (2H, m), 1.64 (9H, s), 1.58-1.48 (2H,m), 1.46 (9H, s); ESI-MS m/z 471 (MH+).

(Step c) Synthesis of4-((6-(1-tert-butyl-3-methyl-1H-pyrazol-5-ylamino)-5-fluoropyridin-2-yl)methyl)-piperidine-4-carbonitrile

The compound (4.11 g, 8.73 mmol) obtained by the aforementioned step bwas dissolved in THF (33 mL), to which MsOH (7.0 mL) was added on awater bath. The solution thus obtained was stirred at room temperaturefor two hours, and the resulting content was poured into 160 mL ofwater. The aqueous solution thus obtained was washed with 50 mL ofisopropyl ether, and 21.5 mL of 5 M sodium hydroxide was added, followedby extraction with ethyl acetate. The ethyl acetate solution thusobtained was washed with saturated brine, and then dried over anhydroussodium sulfate. After filtration, the filtrate was concentrated, wherebythe titled compound (3.09 g, 8.34 mmol) was obtained (yield 96%). Thephysical property values are shown below.

¹H-NMR (CDCl₃) δ: 7.22 (1H, dd, J=10.6, 8.0 Hz), 6.71 (1H, dd, J=8.0,3.2 Hz), 6.25-6.16 (2H, m), 3.02-2.95 (2H, m), 2.91-2.84 (2H, m), 2.83(2H, s), 2.21 (3H, s), 1.90-1.83 (2H, m), 1.61 (9H, s), 1.59-1.49 (2H,m); ESI-MS m/z 371 (MH+).

(Step d) Synthesis of4-((6-(1-tert-butyl-3-methyl-1H-pyrazol-5-ylamino)-5-fluoropyridin-2-yl)methyl)-1-(2,3-dichlorobenzoyl)piperidine-4-carbonitrile

To a mixture of the compound (3.65 g, 9.85 mmol) obtained by theaforementioned step c, 2,3-dichlorobenzoic acid (2.05 g, 10.8 mmol), and1-hydroxybenzotriazole monohydrate (1.80 g, 13.3 mmol), 25 mL ofacetonitrile was added, and then WSC hydrochloride (2.05 g, 10.7 mmol)was added. The resulting reaction mixture was stirred at roomtemperature overnight. Then, 30 mL of 1 M sodium hydroxide was added,followed by stirring for 15 minutes. The mixture thus obtained wasextracted with ethyl acetate. The resulting ethyl acetate layer waswashed sequentially with water, 1 M hydrochloric acid, water, andsaturated brine. The ethyl acetate solution thus obtained was washedwith anhydrous sodium sulfate, and then filtered and concentrated,whereby the titled compound (5.55 g) was obtained as a white solid(yield 100%). The physical property values are shown below.

ESI-MS m/z 543, 545 (MH+).

(Step e) Synthesis of Compound 1

The compound (524 mg, 0.964 mmol) obtained by the aforementioned step dwas dissolved in 3 mL of 1,4-dioxane, and 3 mL of 5 M hydrochloric acidwas then added. The resulting solution was heated at 150° C. for 10minutes in a microwave reaction apparatus. The resulting reactionmixture was concentrated under reduced pressure, and the residue thusobtained was dissolved in chloroform, followed by washing with saturatedbrine. The chloroform solution thus obtained was dried over anhydroussodium sulfate, filtered, and then concentrated under reduced pressure.The resulting residue was purified by silica gel column chromatography(chloroform/methanol=100/0 to 90/10), and the solid thus obtained wasreprecipitated in ethanol-ethyl acetate, whereby the titled compound(290 mg, 0.573 mmol) was obtained as a white solid (yield 59%). Thephysical property values are shown in Table 9.

Examples 2 to 13

Using the raw materials listed in Tables 1 to 3, compounds of Examples 2to 13 were synthesized according to the method of Example 1. Thephysical property values are shown in Tables 9 to 17.

TABLE 1 Example Compound name Raw material 1 Raw material 2 Raw material3 1 1-(2,3-dichlorobenzoyl)-4-((5- fluoro-6-(5-methyl-1H-pyrazol-3-ylamino)pyridin-2-yl)methyl) piperidine-4-carboxylic acid (compound 1)

2 1-(2-fluoro-3- trifluoromethyl)benzoyl)-4-((5-fluoro-6-(5-methyl-1H-pyrazol-3- ylamino)pyridin-2-yl)methyl)piperidine-4-carboxylic acid (compound 2)

3 1-(3-chlorobenzoyl)-4-((5-fluoro- 6-(5-methyl-1H-pyrazol-3-ylamino)pyridin-2-yl)methyl) piperidine-4-carboxylic acid (compound 3)

4 1-(2,3-difluorobenzoyl)-4-((5- fluoro-6-(5-methyl-1H-pyrazol-3-ylamino)pyridin-2-yl)methyl) piperidine-4-carboxylic acid (compound 4)

5 1-(2-fluoro-3-methoxybenzoyl)-4- ((5-fluoro-6-(5-methyl-1H-pyrazol-3-ylamino)pyridin-2-yl) methyl)piperidine-4-carboxylic acid(compound 5)

6 1-(2-chlorobenzoyl)-4-((5-fluoro- 6-(5-methyl-1H-pyrazol-3-ylamino)pyridin-2-yl)methyl) piperidine-4-carboxylic acid (compound 6)

TABLE 2 Example Compound name Raw material 1 Raw material 2 Raw material3 7 1-(2-chloro-3-methylbenzoyl)-4- ((5-fluoro-6-(5-methyl-1H-pyrazol-3-ylamino)pyridin-2-yl)methyl) piperidine-4-carboxylic acid (compound 7)

8 1-(2-chloro-3-fluorobenzoyl)-4- ((5-fluoro-6-(5-methyl-1H-pyrazol-3-ylamino)pyridin-2-yl) methyl)piperidine-4-carboxylic acid(compound 8)

9 1-(2,6-dichlorobenzoyl)-4-((5- fluoro-6-(5-methyl-1H-pyrazol-3-ylamino)pyridin-2-yl)methyl) piperidine-4-carboxylic acid (compound 9)

10 1-(3-chloro-2-fluorobenzoyl)-4- ((5-fluoro-6-(1H-pyrazol-3-ylamino)pyridin-2-yl)methyl) piperidine-4-carboxylic acid (compound 10)

11 1-(3-chloro-2-fluorobenzoyl)-4- ((5-methoxy-6-(5-methyl-1H-pyrazol-3-ylamino)pyridin-2-yl) methyl)piperidine-4-carboxylic acid(compound 11)

TABLE 3 Example Compound name Raw material 1 Raw material 2 Raw material3 12 1-(3-chloro-2-fluorobenzoyl)-4- ((5-chloro-6-(5-methyl-1H-pyrazol-3-ylamino)pyridin-2-yl) methyl)piperidine-4-carboxylic acid(compound 12)

13 1-(3-chloro-2-fluorobenzoyl)-4- ((5-fluoro-6-(5-methyl-1H-pyrazol-3-ylamino)pyridin-2- yl)methyl)piperidine-4- carboxylic acid(compound 13)

Example 14 Synthesis of1-(3-chloro-2-fluorobenzoyl)-4-((5-fluoro-6-(5-methyl-1H-pyrazol-3-ylamino)pyridin-2-yl)methyl)-N-methylpiperidine-4-carboxamide(Compound 14)

To a mixture of the compound 13 (50 mg, 0.1 mmol) obtained in Example13, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (40 mg,0.21 mmol), 1-hydroxybenzotriazole monohydrate (30 mg, 0.22 mmol),methylamine hydrochloride (25 mg, 0.37 mmol), and 1 mL ofdimethylformamide, 0.05 mL of triethylamine was added, followed bystirring at room temperature for 13 hours. To the resulting reactionmixture, water was added, followed by extraction with ethyl acetate. Theresulting extract was dried over anhydrous magnesium sulfate, filtered,and then concentrated. The residue thus obtained was purified by HPLC,whereby the titled compound (41 mg, 0.082 mmol) was obtained as a whitesolid (yield 82%). The physical property values are shown in Tables 9 to17.

Examples 15 to 21

In Examples 15 to 21, using the raw materials specified in Tables 4 to5, compounds were synthesized by a method according to Example 14. Thephysical property values are shown in Tables 9 to 17.

TABLE 4 Example Compound name Raw material 4 141-(3-chloro-2-fluorobenzoyl)-4-((5-fluoro-6-(5- Methylaminemethyl-1H-pyrazol-3-ylamino)pyridin-2-yl) hydrochloridemethyl)-N-methylpiperidine-4-carboxyamide (compound 14) 151-(3-chloro-2-fluorobenzoyl)-4-((5-fluoro-6-(5- Ammoniummethyl-1H-pyrazol-3-ylamino)pyridin-2-yl) chloridemethyl)piperidine-4-carboxyamide (compound 15) 161-(3-chloro-2-fluorobenzoyl)-4-((5-fluoro-6-(5- Dimethylaminemethyl-1H-pyrazol-3-ylamino)pyridin-2-yl) methyl)-N,N-dimethylpiperidine-4- carboxyamide (compound 16) 171-(3-chloro-2-fluorobenzoyl)-N-cyclopropyl-4- Cyclo-((5-fluoro-6-(5-methyl-1H-pyrazol-3-ylamino) propylaminepyridin-2-yl)methyl)piperidine-4- carboxyamide (compound 17) 181-(3-chloro-2-fluorobenzoyl)-N-cyclobutyl-4- Cyclo-((5-fluoro-6-(5-methyl-1H-pyrazol-3-ylamino) butylaminepyridin-2-yl)methyl)piperidine-4- carboxyamide (compound 18) 19azetidin-1-yl(1-(3-chloro-2-fluorobenzoyl)-4- Azetidine((5-fluoro-6-(5-methyl-1H-pyrazol-3-ylamino) hydrochloridepyridin-2-yl)methyl)piperidin-4-yl)methanone (compound 19)

TABLE 5 Example Compound name Raw material 4 20(1-(3-chloro-2-fluorobenzoyl)-4-((5-fluoro-6-(5- Pyrrolidinemethyl-1H-pyrazol-3-ylamino)pyridin-2-yl)methyl)piperidin-4-yl)(pyrrolidin-1-yl) methanone (compound 20) 21(1-(3-chloro-2-fluorobenzoyl)-4-((5-fluoro-6-(5- Isoxazolidinemethyl-1H-pyrazol-3-ylamino)pyridin-2-yl) hydrochloridemethyl)piperidin-4-yl)(isoxazolidin-2-yl) methanone (compound 21)

Example 22 Synthesis of(3-chloro-2-fluorophenyl)(4-((5-fluoro-6-(5-methyl-1H-pyrazol-3-ylamino)pyridin-2-yl)methyl)-4-(5-methyl-1,2,4-oxadiazol-3-yl)piperidin-1-yl)methanone(Compound 22) (Step a) Synthesis oftert-butyl4-((6-(1-tert-butyl-3-methyl-1H-pyrazol-5-ylamino)-5-fluoropyridin-2-yl)methyl)-4-(NT-hydroxycarbamimidoyl)piperidine-1-carboxylate

The compound (50 g) obtained in Example 1 (Step b) was dissolved in 530mL of ethanol at 60° C. The resulting solution was returned to roomtemperature and 65 mL of a 50% aqueous solution of hydroxylamine wasadded, followed by stirring at 60° C. for 46 hours. The resultingreaction solution was added to distilled water, followed by extractionwith ethyl acetate. The resulting organic layer was washed withdistilled water and saturated brine. The resulting solution was driedover sodium sulfate and then concentrated under reduced pressure,whereby the titled compound (53 g, 106 mmol) was obtained (yield 100%).The physical property values are shown below.

ESI-MS m/z 504 (MH+).

(Step b) Synthesis oftert-butyl4-((6-(1-tert-butyl-3-methyl-1H-pyrazol-5-ylamino)-5-fluoropyridin-2-yl)methyl)-4-(5-methyl-1,2,4-oxadiazol-3-yl)piperidine-1-carboxylate

The compound (53 g, 106 mmol) obtained by the aforementioned step a wassuspended in 525 mL of toluene, and 10 mL of acetic anhydride was added,followed by stirring at room temperature for one hour and 20 minutes,and then at 100° C. for 16 hours. To the resulting reaction solution,175 mL of aqueous ammonia, 500 mL of distilled water, and 500 mL ofethyl acetate were sequentially added in an ice bath, followed bywashing with saturated brine. The resulting aqueous layer was extractedwith ethyl acetate, and the resulting organic layer was washed withsaturated brine and dried over sodium sulfate, and then concentratedunder reduced pressure, whereby the titled compound (58 g) was obtainedas a crude purified product. The physical property values are shownbelow.

ESI-MS m/z 528 (MH+).

(Step c) Synthesis ofN-(1-tert-butyl-3-methyl-1H-pyrazol-5-yl)-3-fluoro-6-((4-(5-methyl-1,2,4-oxadiazol-3-yl)piperidin-4-yl)methyl)pyridine-2-amine

The compound (57 g) obtained by the aforementioned step b was dissolvedin 210 mL of acetonitrile, and 27 mL of mesylic acid was added on an icebath, followed by stirring in an ice bath for one hour, and then at roomtemperature for 17 hours. The resulting reaction solution was added to500 mL of distilled water in an ice bath, followed by washing with 500mL of diisopropyl ether. To the resulting aqueous layer, 100 mL of 5 Msodium hydroxide was added in an ice bath, and the aqueous layer wasextracted with ethyl acetate. The resulting organic layer was washedwith saturated brine and dried over sodium sulfate, and thenconcentrated under reduced pressure, whereby the titled compound (44 g,102 mmol) was obtained (yield 91%). The physical property values areshown below.

ESI-MS m/z 428 (MH+).

(Step d) Synthesis of4-((6-(1-tert-butyl-3-methyl-1H-pyrazol-5-ylamino)-5-fluoropyridin-2-yl)methyl)-4-(5-methyl-1,2,4-oxadiazol-3-yl)piperidin-1-yl)(3-chloro-2-fluorophenyl)methanone

The compound (44 g) obtained by the aforementioned step c,3-chloro-2-fluorobenzoic acid (20 g), and 1-hydroxybenzotriazolemonohydrate (21 g) were dissolved in 343 mL of acetonitrile, and in anice bath, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride(22 g) was added, followed by stirring at room temperature for 15 hours.To the resulting reaction solution, 1 M sodium hydroxide (500 mL) wasadded, followed by extraction with ethyl acetate. The resulting organiclayer was washed with distilled water, 1 M hydrochloric acid, distilledwater, and saturated brine, and then dried over sodium sulfate andconcentrated under reduced pressure. The resulting residue wascrystallized from heptane-ethyl acetate, whereby the titled compound (52g, 89 mmol) was obtained (yield 86%). The physical property values areshown below.

ESI-MS m/z 584, 586 (MH+).

(Step e) Synthesis of Compound 22

The compound (2.94 g, 5.03 mmol) obtained by the aforementioned step dwas dissolved in 30 mL of 5 M hydrochloric acid and 20 mL of 2-propanol,followed by heating at 100° C. for two hours. The resulting reactionsolution was cooled on ice, and water and 5 M sodium hydroxide were thenadded to adjust pH to approximately 8, followed by extraction with ethylacetate. The extract thus obtained was washed with water and saturatedbrine, and then dried over sodium sulfate and concentrated under reducedpressure. The resulting residue was purified by silica gel columnchromatography (chloroform/methanol=100/0 to 95/5), whereby the titledcompound (2.26 g, 4.28 mmol) was obtained (yield 85%). The physicalproperty values are shown in Tables 9 to 17.

Examples 23 to 27

In Examples 23 to 27, using the raw materials specified in Tables 6 to7, compounds were synthesized by a method according to Example 22. Thephysical property values are shown in Tables 9 to 17.

TABLE 6 Example Compound name Raw material 5 Raw material 6 Raw material7 22 (3-chloro-2-fluorophenyl)(4-((5- fluoro-6-(5-methyl-1H-pyrazol-3-ylamino)pyridin-2-yl)methyl)-4- (5-methyl-1,2,4-oxadiazol-3-yl)piperidin-1-yl)methanone (compound 22)

Acetic anhydride 23 (2,3-dichlorophenyl)(4-((5-fluoro-6-(5-methyl-1H-pyrazol-3-ylamino) pyridin-2-yl)methyl)-4-(5-methyl-1,2,4-oxadiazol-3-yl)piperidin-1- yl)methanone (compound 23)

Acetic anhydride 24 (3-chloro-2-fluorophenyl)(4-((5-fluoro-6-(5-methyl-1H-pyrazol-3- ylamino)pyridin-2-yl)methyl)-4-(1,2,4-oxadiazol-3-yl)piperidin- 1-yl)methanone (compound 24)

Triethyl orthoformate 25 (3-chloro-2-fluorophenyl)(4-((5-fluoro-6-(5-methyl-1H-pyrazol-3- ylamino)pyridin-2-yl)methyl)-4-(5-(trifluoromethyl)-1,2,4- oxadiazol-3-yl)piperidin-1-yl) methanone(compound 25)

Trifluoroacetic anhydride

TABLE 7 Example Compound name Raw material 5 Raw material 6 Raw material7 26 (3-chloro-2-fluorophenyl)(4-((5- methoxy-6-(5-methyl-1H-pyrazol-3-ylamino)pyridin-2-yl)methyl)- 4-(5-methyl-1,2,4-oxadiazol-3-yl)piperidin-1-yl)methanone (compound 26)

Acetic anhydride 27 (3-chloro-2-fluorophenyl)(4-((5-chloro-6-(5-methyl-1H-pyrazol- 3-ylamino)pyridin-2-yl)methyl)-4-(5-methyl-1,2,4-oxadiazol-3- yl)piperidin-1-yl)methanone (compound 27)

Acetic anhydride

Example 28 Synthesis of(3-chloro-2-fluorophenyl)(4-((5-fluoro-6-(5-methyl-1H-pyrazol-3-ylamino)pyridin-2-yl)methyl)-4-(5-methyl-1,3,4-oxadiazol-3-yl)piperidin-1-yl)methanone(Compound 28) (Step a) Synthesis of tert-butyl2-(1-(3-chloro-2-fluorobenzoyl)-4-((5-fluoro-6-(5-methyl-1H-pyrazol-3-ylamino)pyridin-2-yl)methyl)piperidin-4-carbonyl)hydrazinecarboxylate

The compound 13 (62 mg, 0.13 mmol) obtained in Example 13,tert-butoxycarbonyl hydrazide (25 mg, 0.19 mmol), and1-hydroxybenzotriazole monohydrate (30 mg, 0.22 mmol) were dissolved in3 mL of dimethylformamide, and(1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (41 mg,0.22 mmol) was added, followed by stirring at room temperature for threehours. To the resulting reaction mixture, water was added, and theresulting mixture was extracted with ethyl acetate and then dried overanhydrous magnesium sulfate. The resulting mixture was filtered andconcentrated, and the residue thus obtained was purified by silica gelcolumn chromatography (chloroform/methanol=100/0 to 95/5), whereby thetitled compound (70 mg, 0.12 mmol) was obtained (yield 92%). Thephysical property values are shown below.

ESI-MS m/z 604, 606 (MH+).

(Step b) Synthesis of Compound 28

The compound (70 mg, 0.12 mmol) obtained by the aforementioned step awas dissolved in 4 mL of chloroform, and 2 mL of trifluoroacetic acidwas added, followed by stirring at room temperature for three hours. Theresulting reaction mixture was concentrated, and to the residue,chloroform and a saturated aqueous solution of sodium bicarbonate wereadded for phase separation. The chloroform layer was dried overanhydrous magnesium sulfate, filtered, and concentrated. To the residuethus obtained, 4 mL of toluene and 0.5 mL of ortho ethyl acetate wereadded, followed by stirring while heating at 110° C. for two hours. Tothe resulting reaction solution, water was added at room temperature,followed by extraction with ethyl acetate. The extract thus obtained wasdried over anhydrous magnesium sulfate, and then filtered andconcentrated. The residue thus obtained was purified by silica gelcolumn chromatography (chloroform/methanol=100/0 to 90/10), whereby thetitled compound (39 mg, 0.077 mmol) was obtained (yield 64%). Thephysical property values are shown in Tables 9 to 17.

Example 29 Synthesis of(3-chloro-2-fluorophenyl)(4-((5-fluoro-6-(5-methyl-1H-pyrazol-3-ylamino)pyridin-2-yl)methyl)-4-(3-methyl-1,2,4-oxadiazol-5-yl)piperidin-1-yl)methanone(Compound 29)

To a mixture of the compound 13 (49 mg, 0.10 mmol) obtained in Example13, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (38 mg,0.20 mmol), 1-hydroxybenzotriazole monohydrate (27 mg, 0.20 mmol),acetamidoxime (15 mg, 0.20 mmol), and dimethylformamide (1 mL),diisopropylethylamine (0.07 mL) was added, followed by stirring at roomtemperature for seven hours. To the resulting reaction mixture, waterwas added, followed by extraction with ethyl acetate. The extract thusobtained was dried over anhydrous sodium sulfate, and then filtered andconcentrated. To the crude product thus obtained, 1,4-dioxane (1 mL) wasadded, and the resulting mixture was irradiated at 120° C. for six hourswhile stirring using a microwave reaction apparatus (Biotage Initiator8). After concentration, the residue thus obtained was purified by HPLC,whereby the titled compound (29 mg, 0.055 mmol) was obtained as a lightorange solid (yield 55%). The physical property values are shown inTables 9 to 17.

Comparative Example 1 Synthesis of5-(1-(3-chloro-2-fluorobenzoyl)-4-((6-(1H-pyrazol-3-ylamino)pyridin-2-yl)methyl)piperidin-4-yl)-1,3,4-oxadiazol-2(3H)-one(Comparative Compound 1)

Comparative Compound 1 was synthesized as follows in accordance with themethod described in International Publication No. WO2009/104802. To5-(4-((6-((1-tert-butyl-1H-pyrazol-5-yl)amino)pyridin-2-yl)methyl)-1-(3-chloro-2-fluorobenzoyl)piperidin-4-yl)-1,3,4-oxadiazol-2-(3H)-one(4.45 g, 8.03 mmol), 5 M hydrochloric acid (40 mL) and 2-propanol (40mL) were added, followed by stirring at 100° C. for four hours. To theresulting reaction mixture, 5 M sodium hydroxide (40 mL) was added, andthe resulting solution was separated and extracted with chloroform. Theresulting chloroform extract was dried over anhydrous magnesium sulfate,and then filtered and concentrated. The residue thus obtained waspurified by silica gel column chromatography (chloroform/methanol=100/0to 95/5), and then washed in ethyl acetate while stirring, whereby thetitled compound (1.63 g, 3.29 mmol) was obtained as a light orange solid(yield 41%). The physical property values are shown in Table 18.

Comparative Examples 2 to 5 and 7

In Comparative Examples 2 to 5 and 7, using the raw materials specifiedin Table 8, compounds were synthesized according to a method equivalentto that used in Example 1. The physical property values are shown inTable 18.

TABLE 8 Comparative Example Compound name Raw material 8 Raw material 9Raw material 10 2 1-(3-chloro-2- fluorobenzoyl)-4-((4-cyclopropyl-6-(thiazol- 2-ylamino)pyridin-2- yl)methyl)piperidine-4-carboxylic acid (Comparative Compound 2)

3 1-(3-chloro-2- fluorobenzoyl)-4-((4- cyclopropyl-6-(5-methyl-1H-pyrazol-3- ylamino)pyridin-2- yl)methyl)piperidine-4- carboxylic acid(Comparative Compound 3)

4 1-(3-chloro-2- fluorobenzoyl)-4-((3- methoxy-6-(thiazol-2-ylamino)pyridin-2- yl)methyl)piperidine-4- carboxylic acid (ComparativeCompound 4)

5 4-((6-(1H-pyrazol-3- ylamino)pyridin-2- yl)methyl)-1-(3-chloro-2-fluorobenzoyl)piperidine-4- carboxylic acid (Comparative Compound 5)

7 1-(3-chloro-2- fluorobenzoyl)-4-((3-fluoro- 6-(5-methyl-1H-pyrazol-3-ylamino)pyridin-2- yl)methyl)piperidine-4- carboxylic acid (ComparativeCompound 7)

Comparative Example 6 Synthesis of1-(3-chloro-2-fluorobenzoyl)-4-((5-cyano-6-(5-methyl-1H-pyrazol-3-ylamino)pyridin-2-yl)methyl)piperidine-4-carboxylicAcid (Comparative Compound 6) (Step a) Synthesis of tert-butyl 4-ethyl4-((5-bromo-6-chloropyridin-2-yl)methyl)piperidine-1,4-dicarboxylate

In 21 mL of carbon tetrachloride, 3-bromo-2-chloro-6-methylpyridine (880mg, 4.26 mmol) was dissolved, and N-bromosuccinimide (682 mg, 3.83 mmol)and AIBN (70 mg, 0.426 mmol) were added, followed by stirring at 90° C.for one hour. The resulting reaction solution was concentrated, and theresidue thus obtained was purified by silica gel column chromatography(chloroform/methanol=100/0 to 95/5), whereby3-bromo-6-(bromomethyl)-2-chloropyridine was obtained as a crudepurified product.

In 18 mL of tetrahydrofuran, ethyl N-Boc piperidine carboxylate (1.16mL, 4.72 mmol) was dissolved, and a solution of a lithiumdiisopropylamide/tetrahydrofuran complex in cyclohexane (1.5 M, 3.3 mL,4.96 mmol) was added at −78° C., followed by stirring for 40 minutes.Then, 2 mL of the solution of 3-bromo-6-(bromomethyl)-2-chloropyridinein tetrahydrofuran obtained as above was added dropwise, followed byfurther stirring for 10 minutes. To the resulting reaction solution, asaturated aqueous solution of ammonium chloride was added and thetemperature was raised, and the solution was partitioned between waterand ethyl acetate. The resulting ethyl acetate layer was washed withsaturated brine and dried over anhydrous magnesium sulfate, and thenfiltered and concentrated. The residue thus obtained was purified bysilica gel column chromatography (hexane/ethyl acetate=95/5 to 65/35),whereby the titled compound (592 mg, 1.28 mmol) was obtained (yield27%). The physical property values are shown below.

ESI-MS m/z 461, 463, 465 (MH+).

(Step b) Synthesis of ethyl4-((5-bromo-6-chloropyridin-2-yl)methyl)-1-(3-chloro-2-fluorobenzoyl)piperidine-4-carboxylate

The compound (590 mg, 1.28 mmol) obtained by the aforementioned step awas dissolved in 5 mL of chloroform, and 2 mL of trifluoroacetic acidwas added, followed by stirring at room temperature for one hour. Theresulting reaction solution was concentrated and dissolved in 5 mL ofDMF. Then, 1H-benzo[b][1,2,3]-triazol-1-yl 3-chloro-2-fluorobenzoate(411 mg, 1.41 mmol) and N,N-diisopropylethylamine (0.45 mL, 2.56 mmol)was added at 0° C., followed by stirring for 15 minutes. Water wasadded, and the resulting mixture was extracted with ethyl acetate. Theresulting ethyl acetate layer was washed with water and saturated brine,and dried over anhydrous magnesium sulfate, and then filtered andconcentrated. The residue thus obtained was purified by silica gelcolumn chromatography (hexane/ethyl acetate=90/10 to 50/50), whereby thetitled compound (581 mg, 1.13 mmol) was obtained (yield 88%). Thephysical property values are shown below.

ESI-MS m/z 517, 519, 521 (MH+).

(Step c) Synthesis of4-((5-bromo-6-chloropyridin-2-yl)methyl)-1-(3-chloro-2-fluorobenzoyl)piperidine-4-carboxylicAcid

The compound (310 mg, 0.598 mmol) obtained by the aforementioned step bwas dissolved in 6 mL of ethanol, and 5 M sodium hydroxide (0.96 mL) wasadded, followed by stirring at 80° C. for 1.5 hours. The resultingreaction solution was diluted with water, and 5 M hydrochloric acid wasadded to adjust pH to 1, followed by extraction with ethyl acetate. Theresulting ethyl acetate layer was washed with water and saturated brine,and dried over anhydrous magnesium sulfate, and then filtered andconcentrated. The residue thus obtained was purified by silica gelcolumn chromatography (hexane/ethyl acetate=30/70 to0/100→chloroform/methanol=100/0 to 90/10), whereby the titled compound(259 mg, 0.526 mmol) was obtained (yield 88%). The physical propertyvalues are shown below.

ESI-MS m/z 489, 491, 493 (MH+).

(Step d) Synthesis of Comparative Compound 6

To the compound (80 mg, 0.163 mmol) obtained by the aforementioned stepc and copper cyanide (16 mg, 0.180 mmol), 1 mL of N-methylpyrrolidonewas added, and the resulting mixture was irradiated at 195° C. for 40minutes while stirring using a microwave reaction apparatus (BiotageInitiator 8). The resulting reaction solution was diluted with water and1 M hydrochloric acid was added, followed by extraction with ethylacetate. The resulting ethyl acetate layer was washed with water andsaturated brine, and dried over anhydrous magnesium sulfate, and thenfiltered and concentrated. The residue thus obtained was purified bysilica gel column chromatography (hexane/ethyl acetate=50/50 to0/100→chloroform/methanol=100/0 to 90/10), whereby4-((5-cyano-6-chloropyridin-2-yl)methyl)-1-(3-chloro-2-fluorobenzoyl)piperidine-4-carboxylicacid (9 mg) was obtained as a crude purified product. To the crudepurified product obtained in this step (9 mg),5-amino-1-tert-butyl-3-methylpyrazole (3.5 mg, 0.022 mmol), xantphos(2.4 mg, 0.0041 mmol), Pd₂(dba)₃ (2.0 mg, 0.0023 mmol), and potassiumphosphate (8.7 mg, 0.041 mmol), 0.2 mL of dioxane was added, followed bystirring at 100° C. for 3.5 hours. The resulting reaction solution wasdiluted with water and extracted with ethyl acetate. The resulting ethylacetate layer was washed with water and saturated brine, and dried overanhydrous magnesium sulfate, and then filtered and concentrated. Theresidue thus obtained was purified by silica gel column chromatography(hexane/ethyl acetate=25/75 to 0/100→chloroform/methanol=100/0 to90/10), whereby4-((6-(1-tert-butyl-5-methyl-1H-pyrazol-3-ylamino)-5-cyanopyridin-2-yl)methyl)-1-yl)-1-(3-chloro-2-fluorobenzoyl)piperidine-4-carboxylicacid (4 mg) was obtained as a crude product. The crude product thusobtained (4 mg) was dissolved in 0.5 mL of trifluoroacetic acid and 0.05mL of anisole, followed by stirring while heating at 85° C. for onehour. After concentration, the residue thus obtained was purified byreverse phase HPLC, whereby the titled compound (1 mg, 0.002 mmol) wasobtained (yield 1%). The physical property values are shown in Table 18.

TABLE 9 Example Structural formula Physical property value Example 1

¹H-NMR (DMSO-D₆) δ: 10.05 (1H, brs), 7.70-7.65 (1H, m), 7.59-7.54 (1H,m), 7.46-7.29 (2H, m), 6.72- 6.68 (1H, m), 6.30-6.29 (1H, m), 4.23-4.18(1H, m), 3.26-3.20 (1H, m), 3.09-2.95 (4H, m), 2.27- 2.26 (3H, m),2.05-2.00 (1H, m), 1.90-1.83 (1H, m), 1.67-1.49 (2 H, m); ESI-MS m/z506, 508 (MH+). Example 2

¹H-NMR (DMSO-D₆) δ: 10.05 (1H, brs), 7.86 (1H, t, J = 7.7 Hz), 7.75 (1H,t, J = 7.7 Hz), 7.57 (1H, dd, J = 11.1, 8.2 Hz), 7.48 (1H, t, J = 7.7Hz), 6.70 (1H, dd, J = 8.2, 2.8 Hz), 6.29 (1H, s), 4.20 (1H, d, J = 13.7Hz), 3.36 (1H, d, J = 13.7 Hz), 3.11-2.98 (4H, m), 2.25 (3H, s), 2.02(1H, d, J = 13.7 Hz), 1.91-1.87 (1H, m), 1.64-1.50 (2H, m); ESI-MS m/z524 (MH+). Example 3

¹H-NMR (CDCl₃) δ: 7.38-7.00 (5H, m), 6.70-6.58 (1H, m), 5.68 (1H, s),4.60-4.45 (1H, m), 3.40-3.09 (4H, m), 3.04-2.89 (1H, m), 2.40- 2.10 (2H,m), 2.24 (3H, s), 1.71- 1.42 (2H, m); ESI-MS m/z 472, 474 (MH+).

TABLE 10 Example Structural formula Physical property value Example 4

¹H-NMR (CDCl₃) δ: 7.30-6.95 (4H, m), 6.68-6.60 (1H, m), 5.69 (1H, s),4.64-4.55 (1H, m), 3.42- 3.15 (4H, m), 3.03-2.90 (1H, m), 2.50-2.30 (1H,m), 2.28-2.16 (1H, m), 2.23 (3H, s), 1.58-1.40 (2H, m); ESI-MS m/z 474(MH+). Example 5

¹H-NMR (DMSO-D₆) δ: 9.23 (1H, brs), 7.73-7.65 (1H, m), 7.58- 7.46 (2H,m), 7.23-7.13 (1H, m), 6.83-6.79 (1H, m), 6.60 (1H, s), 4.51-4.40 (1H,m), 4.17 (3H, s), 3.75-3.36 (1H, m), 3.52- 3.30 (2H, m), 3.20 (2H, s),2.46 (3H, s), 2.39-2.27 (1H, m), 2.23-2.12 (1H, m), 1.97-1.78 (2H, m);ESI-MS m/z 486 (MH+). Example 6

¹H-NMR (CDCl₃) δ: 7.38-7.02 (4H, m), 6.68-6.59 (1H, m), 5.68 (1H, s),4.66-4.56 (1H, m), 3.33- 3.09 (4H, m), 3.03-2.91 (1H, m), 2.40-2.25 (1H,m), 2.24 (3H, s), 2.20-2.10 (1H, m), 1.62- 1.40 (2H, m); ESI-MS m/z 472,474 (MH+). Example 7

¹H-NMR (DMSO-D₆) δ: 9.24 (1H, brs), 7.75-7.43 (4H, m), 6.87- 6.79 (1H,m), 6.60 (1H, s), 4.54- 4.41 (1H, m), 3.71-3.29 (3H, m), 3.20 (2H, s),2.66 (3H, s), 2.46 (3H, s), 2.39-2.28 (1H, m), 2.22-2.12 (1H, m),2.00-1.80 (2H, m); ESI-MS m/z 486, 488 (MH+).

TABLE 11 Example Structural formula Physical property value Example 8

¹H-NMR (DMSO-D₆) δ: 9.23 (1H, brs), 7.82-7.63 (3H, m), 7.60- 7.48 (1H,m), 6.85-6.79 (1H, m), 6.66-6.56 (1H, m), 4.56-4.44 (1H, m), 3.72-3.29(3H, m), 3.24-3.12 (2H, m), 2.47 (3H, s), 2.41-2.29 (1H, m), 2.24- 2.12(1H, m), 2.01-1.80 (2H, m); ESI-MS m/z 490, 492 (MH+). Example 9

¹H-NMR (DMSO-D₆) δ: 9.23 (1H, brs), 7.89-7.63 (4H, m), 6.85- 6.79 (1H,m), 6.61 (1H, s), 4.58- 4.48 (1H, m), 3.72-3.30 (3H, m), 3.26-3.14 (2H,m), 2.47 (3H, s), 2.41-2.30 (1H, m), 2.27- 2.16 (1H, m), 2.01-1.85 (2H,m); ESI-MS m/z 506, 508 (MH+) . Example 10

¹H-NMR (DMSO-D₆) δ 9.07 (1H, brs), 7.65 (1H, td, J = 7.8, 2.0 Hz), 7.49(1H, d, J = 2.0 Hz), 7.41- 7.35 (2H, m), 7.29 (1H, t, J = 7.8 Hz),6.53-6.51 (2H, m), 4.19 (1H, d, J = 13.7 Hz), 3.34 (1H, d, J = 13.7 Hz),3.07-3.02 (2H, m), 2.90 (1H, d, J = 13.4 Hz), 2.87 (1H, d, J = 13.4 Hz),2.03 (1H, d, J = 13.4 Hz), 1.88 (1H, d, J = 13.4 Hz), 1.61-1.48 (2H, m);ESI-MS m/z 476, 478 (MH+). Example 11

¹H-NMR (DMSO-D₆) δ: 10.68 (1H, brs), 7.67 (1H, t, J = 7.6 Hz), 7.51 (1H,d, J = 8.0 Hz), 7.37 (1H, t, J = 6.7 Hz), 7.30 (1H, t, J = 7.6 Hz), 6.85(1H, d, J = 8.0 Hz), 6.14 (1H, s), 4.32-4.20 (1H, m), 3.97 (3H, s),3.43-3.32 (1H, m), 3.15-2.96 (4H, m), 2.27 (3H, s), 2.12-2.03 (1H, m),1.99-1.87 (1H, m), 1.70-1.45 (2H, m); ESI-MS m/z 502, 504 (MH+).

TABLE 12 Example Structural formula Physical property value Example 12

¹H-NMR (CDCl₃) δ: 7.55 (1H, d, J = 8.7 Hz), 7.42-7.35 (1H, m), 7.34 (1H,s), 7.25-7.16 (2H, m), 6.70- 6.61 (1H, m), 5.67 (1H, s), 4.61- 4.53 (1H,m), 3.40-3.09 (4H, m), 3.05-2.94 (1H, m), 2.37-2.10 (2H, m), 2.25 (3H,s), 1.61-1.40 (2H, m); ESI-MS m/z 506, 508 (MH+). Example 13

¹H-NMR (DMSO-D₆) δ 10.13 (1H, br s), 7.68-7.64 (1H, m), 7.58 (1H, dd, J= 11.0 Hz, 8.0 Hz), 7.38-7.35 (1H, m), 7.29 (1H, t, J = 8.0 Hz),6.73-6.70 (1H, m), 6.29 (1H, s), 4.21-4.18 (1H, m), 3.38-3.34 (1H, m),3.09-2.99 (4H, m), 2.27 (3H, s), 2.03-1.99 (1H, m), 1.89- 1.87 (1H, m),1.63-1.49 (2H, m); ESI-MS m/z 490, 492 (MH+). Example 14

¹H-NMR (DMSO-D₆) δ: 11.79 (1H, s), 8.68 (0.5H, s), 7.68-7.61 (2H, m),7.36-7.27 (3H, m), 6.41 (1.5H, s), 4.06-4.01 (1H, m), 3.32 (3H, s),3.30-3.28 (1H, m), 3.20-3.16 (1H, m), 3.09-3.03 (1H, m), 2.87-2.82 (2H,m), 2.13 (3H, s), 2.05-2.01 (1H, m), 1.91-1.87 (1H, m), 1.61- 1.49 (2H,m); ESI-MS m/z 503, 505 (MH+).

TABLE 13 Example Structural formula Physical property value Example 15

¹H-NMR (DMSO-D₆) δ: 11.79 (1H, brs), 8.66 (0.5H, brs), 7.65 (1H, td, J =7.7, 1.6 Hz), 7.37-7.26 (4H, m), 6.98 (1H, s), 6.48 (1H, brs), 6.41(0.5H, brs), 4.09- 4.04 (1H, m), 3.31-3.28 (1H, m), 3.21-3.18 (1H, m),3.12- 3.07 (1H, m), 2.86-2.83 (2H, m), 2.13 (3H, s), 2.06-2.03 (1H, m),1.92-1.89 (1H, m), 1.61-1.48 (2H, m); ESI-MS m/z 489, 491 (MH+). Example16

¹H-NMR (DMSO-D₆) δ: 11.74 (1H, brs), 7.65 (1H, td, J = 7.7, 1.6 Hz),7.37-7.26 (3H, m), 6.49-6.47 (1H, m), 6.17 (1H, brs), 4.13- 4.10 (1H,m), 3.30-3.28 (1H, m), 3.15-2.86 (10H, m), 2.23- 2.19 (1H, m), 2.15 (3H,s), 2.11- 2.07 (1H, m), 1.66-1.50 (2H, m); ESI-MS m/z 517, 519 (MH+).Example 17

¹H-NMR (DMSO-D₆) δ: 11.81 (1H, brs), 7.68-7.63 (2H, m), 7.38- 7.26 (3H,m), 6.41 (1H, brs), 4.08-4.04 (1H, m), 3.29-3.27 (1H, m), 3.14-3.02 (2H,m), 2.84 (2H, brs), 2.51-2.46 (1H, m), 2.12 (3H, s), 2.09-2.05 (1H, m),1.95-1.92 (1H, m), 1.60- 1.47 (2H, m), 0.53-0.48 (2H, m), 0.34-0.31 (2H,m); ESI-MS m/z 529, 531 (MH+).

TABLE 14 Example Structural formula Physical property value Example 18

¹H-NMR (DMSO-D₆) δ: 11.80 (1H, brs), 8.67 (0.5H, brs), 7.71 (1H, d, J =7.6 Hz), 7.66 (1H, td, J = 7.6, 1.5 Hz), 7.39-7.29 (2H, m), 7.28 (1H, t,J = 7.8 Hz), 6.38 (1.5H, brs), 4.17-4.15 (1H, brm), 4.06-4.03 (1H, brm),3.31-3.29 (1H, m), 3.19-3.16 (1H, m), 3.07-3.02 (1H, m), 2.83 (2H, brs),2.13 (3H, s), 1.94-1.93 (1H, m), 2.07-2.05 (1H, m), 2.00-1.96 (2H, m),1.87-1.78 (2H, m), 1.63-1.49 (4H, m); ESI-MS m/z 543, 545 (MH+). Example19

¹H-NMR (DMSO-D₆) δ: 8.99 (1H, brs), 7.66 (1H, td, J = 7.7, 1.5 Hz),7.42-7.35 (2H, m), 7.29 (1H, t, J = 7.7 Hz), 6.54-6.51 (1H, m), 6.27(1H, brs), 4.14-4.10 (1H, m), 3.87-3.72 (4H, m), 3.36- 3.34 (1H, m),3.13-3.00 (2H, m), 2.82 (2H, s), 2.15 (3H, s), 2.08-2.02 (1H, m),1.95-1.88 (3H, m), 1.56-1.45 (2H, m); ESI-MS m/z 529, 531 (MH+). Example20

¹H-NMR (DMSO-D₆) δ: 11.80 (1H, brs), 8.69 (1H, brs), 7.65 (1H, td, J =7.7, 1.7 Hz), 7.38-7.29 (2H, m), 7.29 (1H, t, J = 7.7 Hz), 6.57-6.36(2H, m), 4.14-4.10 (1H, m), 3.34-3.27 (5H, m), 3.15- 3.02 (2H, m), 2.87(2H, brs), 2.26-2.22 (1H, m), 2.12-2.10 (1H, m), 2.15 (3H, s), 1.65-1.53(6H, m); ESI-MS m/z 543, 545 (MH+).

TABLE 15 Example Structural formula Physical property value Example 21

¹H-NMR (CDCl₃) δ: 7.42 (1H, ddd, J = 8.0, 7.1, 1.7 Hz), 7.23 (1H, brs),7.17 (1H, dd, J = 10.5, 8.0 Hz), 7.12 (1H, dd, J = 8.0, 7.1 Hz), 7.07(1H, brs), 6.53-6.50 (1H, m), 5.85 (1H, s), 4.48-4.45 (1H, m), 4.13-4.07(2H, m), 3.84-3.73 (2H, m), 3.39-3.37 (2H, brm), 3.24-3.12 (2H, m), 3.03(1H, d, J = 13.7 Hz), 2.52 (1H, d, J = 13.7 Hz), 2.37-2.29 (3H, m), 2.26(3H, s), 1.67-1.42 (2H, m); ESI-MS m/z 545, 547 (MH+). Example 22

¹H-NMR (CDCl₃) δ: 7.45 (1H, ddd, J = 8.2, 6.6, 1.3 Hz), 7.34-7.15 (1H,m), 7.15 (1H, t, J = 7.7 Hz), 7.14 (1H, dd, J = 10.6, 8.1 Hz), 7.03 (1H,brs), 6.42-6.31 (1H, m), 5.98 (1H, s), 4.63-4.52 (1H, m), 3.50-3.38 (1H,m), 3.17 (1H, brs), 3.10 (1H, d, J = 13.2 Hz), 3.05 (1H, d, J = 13.2Hz), 3.02-2.89 (1H, m), 2.53-2.39 (1H, m), 2.51 (3H, s), 2.37-2.26 (1H,m), 2.31 (3H, s), 1.96-1.66 (2H, m); ESI-MS m/z 528, 530 (MH+). Example23

¹H-NMR (CDCl₃) δ 7.50-7.47 (1H, m), 7.28-7.23 (1H, m), 7.16- 7.08 (2H,m), 6.38-6.33 (1H, m), 6.04-5.99 (1H, m), 4.60 (1H, d, J = 13.9 Hz),3.37-3.29 (1H, m), 3.19-3.03 (3H, m), 3.01- 2.91 (1H, m), 2.52-2.43 (4H,m), 2.32-2.27 (4H, m), 1.98- 1.84 (2H, m); ESI-MS m/z 544, 546 (MH+).

TABLE 16 Example Structural formula Physical property value Example 24

¹H-NMR (CDCl₃) δ: 8.73 (1H, s), 7.46 (1H, t, J = 7.5 Hz), 7.32- 7.10(3H, m), 7.00 (1H, s), 6.37 (1H, brs), 5.92 (1H, s), 4.63-4.52 (1H, m),3.52-3.40 (1H, m), 3.27-2.88 (4H, m), 2.54-2.43 (1H, m), 2.40-2.30 (1H,m), 2.31 (3H, s), 2.02-1.68 (2H, m); ESI- MS m/z 514, 516 (MH+). Example25

¹H-NMR (CDCl₃) δ: 7.46 (1H, ddd, J = 8.3, 6.7, 1.2 Hz), 7.26- 7.17 (2H,m), 7.16 (1H, t, J = 7.7 Hz), 7.12 (1H, dd, J = 10.6, 8.0 Hz), 6.28 (1H,dd, J = 7.0, 2.4 Hz), 6.15 (1H, brs), 4.64- 4.50 (1H, m), 3.60-3.43 (1H,m), 3.24-2.92 (4H, m), 2.56-2.45 (1H, m), 2.42-2.34 (1H, m), 2.32 (3H,s), 2.09-1.81 (2H, m); ESI- MS m/z 582, 584 (MH+). Example 26

¹H-NMR (CDCl₃) δ: 7.44 (1H, ddd, J = 8.3, 6.5, 1.0 Hz), 7.30-7.10 (3H,m), 6.86 (1H, d, J = 8.1 Hz), 6.39 (1H, brs), 5.72 (1H, s), 4.64-4.49(1H, m), 3.86 (3H, s), 3.49-3.35 (1H, m), 3.28- 2.85 (2H, m), 3.06 (2H,s), 2.52 (3H, s), 2.49-2.39 (1H, m), 2.37- 2.28 (1H, m), 2.27 (3H, s),1.94- 1.64 (2H, m); ESI-MS m/z 540, 542 (MH+). Example 27

¹H-NMR (CDCl₃) δ 7.50-7.39 (2H, m), 7.36-7.09 (2H, m), 6.42- 6.30 (1H,m), 6.04-5.89 (1H, m), 4.61-4.50 (1H, m), 3.50- 3.39 (1H, m), 3.29-2.88(4H, m), 2.52-2.38 (4H, m), 2.36-2.20 (4H, m), 1.95-1.78 (2H, m); ESI-MS m/z 544, 546 (MH+).

TABLE 17 Example Structural formula Physical property value Example 28

¹H-NMR (CDCl₃) δ: 7.46 (1H, ddd, J = 8.3, 6.7, 1.3 Hz), 7.33-7.20 (2H,brm), 7.16 (1H, t, J = 7.5 Hz), 7.14 (1H, dd, J = 10.6, 7.9 Hz), 6.34(1H, dd, J = 7.8, 2.1 Hz), 6.07 (1H, s), 4.66-4.57 (1H, m), 3.53-3.45(1H, m), 3.20 (1H, brs), 3.09 (1H, d, J = 13.4 Hz), 3.05 (1H, d, J =13.4 Hz), 3.00-2.84 (1H, m), 2.52-2.43 (1H, m), 2.43-2.34 (1H, m), 2.38(3H, s), 2.32 (3H, s), 2.01-1.66 (2H, m); ESI-MS m/z 528, 530 (MH+).Example 29

¹H-NMR (CDCl₃) δ: 7.45 (1H, ddd, J = 8.3, 6.7, 1.3 Hz), 7.34-7.16 (2H,m), 7.14 (1H, t, J = 7.8 Hz), 7.11 (1H, dd, J = 10.7, 7.8 Hz), 6.34-6.20(1H, m), 6.11 (1H, s), 4.66-4.50 (1H, m), 3.58-3.40 (1H, m), 3.20-3.02(1H, m), 3.10 (2H, s), 3.01-2.88 (1H, m), 2.58-2.45 (1H, m), 2.43-2.30(1H, m), 2.32 (3H, s), 2.26 (3H, s), 2.03-1.65 (2H, brm); ESI-MS m/z528, 530 (MH+).

TABLE 18 Example Structural formula Physical property value ComparativeExample 1

1H-NMR (CDCl₃) δ: 7.60 (1H, m), 7.55-7.20 (4H, m), 6.81 (1H, m), 6.58-6.20 (2H, m), 4.45 (1H, m), 3.48 (1H, m), 3.35-3.12 (2H, m), 3.04 (2H,s), 2.28 (1H, m), 2.15 (1H, m), 1.94-1.77 (2H, m); ESI-MS m/z 498, 500(MH+). Comparative Example 2

1H-NMR (DMSO-D₆) δ 7.68-7.60 (1H, m), 7.58-7.43 (1H, m), 7.40-7.00 (3H,m), 6.72 (1H, s), 6.50 (1H, s), 4.32-4.20 (1H, m), 3.38-3.28 (1H, m),3.09-2.88 (3H, m), 2.13-1.40 (5H, m), 1.13-1.00 (2H, m), 0.84-0.65 (2H,m); ESI-MS m/z 515, 517 (MH+). Comparative Example 3

¹H-NMR (CDCl₃) δ: 7.50-7.10 (3H, m), 6.55-6.41 (1H, m), 6.25 (1H, s),5.70-5.50 (1H, m), 4.45- 4.20 (1H, m), 3.40-3.09 (5H, m), 2.32-1.95 (5H,m), 1.85-1.20 (3H, m), 1.15-1.00 (2H, m), 0.87-0.65 (2H, m); ESI-MS m/z512, 514 (MH+). Comparative Example 4

¹H-NMR (CDCl₃) δ: 7.48 (1H, t, J = 7.4 Hz), 7.24-7.16 (2H, m), 7.10 (1H,d, J = 8.8 Hz), 6.99 (1H, s), 6.41 (1H, s), 6.19 (1H, brs), 4.73-4.55(1H, m), 3.83 (3H, s), 3.58-3.44 (1H, m), 3.39- 2.96 (4H, m), 2.49-2.14(2H, m), 1.89-1.44 (2H, m); ESI-MS m/z 505, 507 (MH+) ComparativeExample 5

¹H-NMR (CD3OD) δ: 7.90-7.85 (1H, m), 7.69 (1H, brs), 7.10-7.02 (1H, m),6.95-6.87 (1H, m), 6.11 (1H, brs), 4.53-4.42 (1H, m), 3.55- 3.45 (1H,m), 3.30-3.05 (4H, m), 2.33-2.26 (1H, m), 2.20-2.08 (1H, m), 1.80-1.58(2H, m); ESI-MS m/z 458, 460 (MH+). Comparative Example 6

¹H-NMR (CDCl₃) δ: 9.03 (1H, d, J = 8.6 Hz), 7.49 (1H, t, J = 7.5 Hz),7.31-7.24 (1H, m), 7.28 (1H, d, J = 8.6 Hz), 7.20 (1H, t, J = 7.5 Hz),6.14 (1H, s), 4.56-4.37 (1H, m), 3.50-3.41 (1H, m), 3.23 (2H, s),3.11-2.90 (2H, m), 2.53 (3H, s), 2.36-2.26 (2H, m), 2.22-2.13 (2H, m),1.80-1.46 (2H, m); ESI-MS m/z 497, 499 (MH+). Comparative Example 7

¹H-NMR (CDCl₃) δ: 7.55-7.42 (1H, m), 7.35-7.10 (3H, m), 6.57 (1H, dd, J= 9.2, 2.9 Hz), 5.52 (1H, s), 4.50-4.40 (1H, m), 3.45- 3.04 (5H, m),2.26-2.19 (1H, m), 2.23 (3H, s), 2.15-2.05 (1H, m), 1.71-1.40 (2H, m);ESI-MS m/z 490, 492 (MH+).

Test Example 1: Evaluation of Inhibitory Activities on Aurora A andAurora B

The inhibitory activity of a test compound on aurora A and aurora B wasmeasured in accordance with the following method. As a control compound,MLN8237, which is under clinical development as an aurora A-selectiveinhibitor, was used.

1) Purification of Aurora A Protein

The cDNA encoding human aurora A having a N-term fused histidine tag wasinserted into an expression vector and this protein was expressed at ahigh level in the E. coli BL21-CodonPlus (DE3)-RIL strain. The E. coliwere collected and solubilized, and histidine tag-fused human aurora Aprotein was extracted by adsorption to a nickel chelate column and theneluted with imidazole from the column. The active fraction was desaltedwith a desalting column, whereby a purified enzyme was obtained.

2) Measurement of the Inhibitory Activity on Aurora A

For in vitro method for measuring the inhibitory activity of theaforementioned compounds on the aurora A kinase activity was carried outreferring to the method described in JP-A-2008-81492. As the first stepof measuring the inhibitory activity of the compound, the test compoundwas serially diluted with dimethyl sulfoxide (DMSO). Subsequently,purified human aurora A protein, FL-Peptide 21 (Caliper Life Sciences,Inc., a final concentration of 100 nM), ATP (a final concentration of 5μM), and the solution of the compound of the present invention in DMSO(a final DMSO concentration of 5%) were added to a reaction buffer [50mM Tris-hydrochloric acid buffer (pH 7.4), 15 mM magnesium acetate, and0.2 mM ethylenediamine-N, N, N′, N′-tetraacetic acid (EDTA)]. Then, theresulting mixture was incubated at 25° C. for 50 minutes to carry outkinase reactions. Then, the IMAP® Progressive Binding Reagent diluted500-fold with the IMAP® Progressive Binding Buffer A (the product ofMolecular Devices, LLC.) was added thereto to terminate the kinasereaction. After leaving the resulting product to stand in the dark atroom temperature for 120 minutes, the amount of phosphorylation wasdetermined from the degree of fluorescence polarization as measured bythe PHERAstar (BMG LABTECH, excitation wavelength of 485 nm, detectionwavelength of 520 nm). Then, the concentration of the compound at whichthe phosphorylation reaction can be inhibited by 50% was defined as theIC₅₀ value (nM), and the results were shown in Table 19.

3) Measurement of the Aurora B Kinase Activity

The in vitro method for measuring the inhibitory activity of the testcompound on the aurora B kinase activity was performed in a similarmanner as the above method for aurora A, and purified recombinant humanaurora B protein was purchased from Carna Biosciences, Inc. The reactionbuffer has the following composition: 20 mM HEPES (pH 7.4), 2 mM DTT,0.01% Tween-20, magnesium chloride at a final concentration of 1 mM, andATP at a final concentration of 40 μM, and the incubation time was 60minutes. The concentration of the compound at which the phosphorylationreaction can be inhibited by 50% was defined as the IC₅₀ value (nM) andthe results were shown in Table 19.

TABLE 19 Aurora A Aurora B Example No. IC₅₀ (nM) IC₅₀ (nM)  1 0.4 140  20.5 340 11 0.6 930 12 0.4 260 13 0.4 180 14 0.9 460 17 0.9 900 19 1.0680 22 0.7 380 24 0.5 190 28 0.7 450 29 0.8 390 Comparative 220 320Example 6 MLN8237 0.6 90

As a result, it was confirmed that the compounds of the presentinvention exhibited a higher inhibitory activity on aurora A and a lowerinhibitory activity on aurora B even in comparison with MLN8237, whichis the control compound, thereby exhibiting selectivity for aurora A. Incontrast, Comparative Example 6 did not exhibit either inhibitoryactivity on aurora A or selectivity for aurora A. From the aboveresults, it was suggested that incorporation of a specific substituentinto the specific position (a halogen atom or a C₁-C₆ alkoxy group atR₂) on the pyridine ring in the structure of the compound of the presentinvention represented by the general formula (I) could impart not only ahigh inhibitory activity on aurora A, but also aurora A selectivity.

Test Example 2: Evaluation of the Inhibitory Effect on CellProliferation

Cells of the human-derived stomach cancer cell line SNU-16 were eachroutinely subcultured in the 10% fetal bovine serum (FBS)-containingRPMI-1640 medium and 10% FBS-containing Dulbecco's Modified Eagle Medium(DMEM) in order to maintain cell density of no more than 80%. In orderto initiate a test for the inhibitory activity on cell proliferation,cells were each suspended in the aforementioned medium and seeded ineach well of a 96-well flat bottom plate (black plate with a transparentbottom) at 2,500 or 3,000 cells per well. The cells were then culturedfor one day at 37° C. in an incubator containing 5% carbon dioxide gas.The next day, the compound of the present invention and the subjectcompound were dissolved in DMSO, and using DMSO, the test compounds wereserially diluted to a concentration of 200 times the finalconcentration. The solution of the test compounds in DMSO was dilutedwith the medium used for culturing, and then added to each well of thecell culture plate at a final DMSO concentration of 0.5%. The cells werecultured for 72 hours at 37° C. in an incubator containing 5% carbondioxide gas. The cells were counted at the time of addition of the testcompounds and 72 hours after culturing using the CellTiter-GloLuminescent Cell Viability Assay kit (the product of Promega) based onthe protocol recommended by Promega. The reagent included in the kit wasadded to each plate, followed by stirring, and the plates were left tostand at room temperature for 10 minutes. Upon completion of thereaction, the luminescence signal was measured using a microplatereader.

The cell proliferation inhibitory rate was calculated from the followingformula and the concentration of the test compound at which the cellproliferation was inhibited by 50% (GI₅₀ (nM)) was determined. Theresults were shown in Table 20.

Cell proliferation inhibitory rate (%)=(C−T)/(C−C0)×100

T: Luminescence signal in a well with the addition of the test compoundC: Luminescence signal in a well without the addition of the testcompoundC0: Luminescence signal in a well measured before the addition of thecompound

TABLE 20 Example No. GI₅₀ (nM) 1 120 13 60 22 970

As a result, the compound of the present invention exhibited theinhibitory effect on cell proliferation, and thus was suggested to beuseful as an anti-tumor agent.

Test Example 3: Evaluation of Oral Absorbability

The test compound was suspended or dissolved in 0.5% HPMC and orallyadministered to a BALB/cA mouse. The blood was drawn from theretro-orbital venous plexus 0.5, 1, 2, 4, and 6 hours after oraladministration to obtain plasma. The concentration of the compound inthe plasma thus obtained was measured by LCMS and the oral absorbabilitywas evaluated.

As a result, after oral administration, adequate plasma concentrationswere observed with the compounds of Examples 1, 11, 12, 13, and 22,which are the compounds of the present invention, showing favorable oralabsorbability. Meanwhile, Comparative Examples 2 to 5 and 7 did notexhibit adequate oral absorbability (AUC_(0-6hr) was less than ⅛ of thatof the compounds of the present invention). Therefore, it was considereddifficult to incorporate those compounds in orally administeredpreparations as the active ingredient, and thus no clinical effect wouldbe expected from oral administration. From the above results, it wasrevealed that incorporation of a halogen atom or a C₁-C₆ alkoxy group atR₂ on the pyridine ring in the structure of the compound of the presentinvention represented by the general formula (I) imparted high oralabsorbability.

Test Example 4: Evaluation of the Anti-Tumor Effect (1)

The luciferase-introduced human uterine cervical cancer cell line(Hela-Luc) was subcutaneously transplanted into a nude mouse, and whenthe volume of the grafted tumor reached 100 to 200 mm³, five mice in onegroup were grouped into the single drug administration group andcombinational administration group by a randomized stratification methodso as to achieve a uniform tumor volume in each group (day 1). In thesingle drug administration groups, Group 1: paclitaxel (30 mg/kg) wasintravenously administered on day 1, Group 2: the compound of thepresent invention (Compound 1) (60 mg/kg) was orally administered twicea day on day 2 and day 3, and Group 3: Comparative Compound 1 (60 mg/kg)was orally administered twice a day on day 2 and day 3. In thecombination administration groups, Group 4: paclitaxel (30 mg/kg) wasintravenously administered on day 1 and Compound 1 (60 mg/kg) was orallyadministered twice a day on day 2 and day 3, and Group 5: paclitaxel (30mg/kg) was intravenously administered on day 1 and Comparative Compound1 (60 mg/kg) was orally administered twice a day on day 2 and day 3. Inorder to compare the anti-tumor effect brought about by drugadministration, setting the tumor volume at the time of grouping at 1,the relative tumor volume (RTV) was determined in accordance with thefollowing formula as a rate of tumor proliferation.

RTV=(Tumor volume on the day of measurement of tumor volume)/(Tumorvolume at the time of grouping)

In Table 21, the average RTV values of the control and single drugadministration groups (Groups 2 and 3) on the 23^(rd) day after groupingare shown, and the average RTV values of the paclitaxel-onlyadministered group (Group 1) and the combination administration groups(Groups 4 and 5) on the 23^(rd) and 46^(th) days after grouping areshown.

Also, the disease control rate (DCR) described in, for example, J. Clin.Oncol., 29 (31), pp. 4129 to 4136, (2010) was also used as the index ofthe anti-tumor effect brought about by drug administration. DCR wasdefined as the ratio of individuals in which RTV does not exceed 1 onthe final day of tumor volume measurement (day 46). DCR was obtained inaccordance with the following formula and the results were shown inTable 21.

DRC (%)=[(Number of individuals in which RTV does not exceed 1 on thefinal day of tumor volume measurement)/(Number of mice survived on thefinal day)]×100

Meanwhile, as the index of systemic toxicity caused by drugadministration, the body weight change (BWC) was used. BWC wascalculated in accordance with the following formula and the average BWCvalues were shown in Table 21.

BWC (%)=([(Body weight of mouse on the day of body weightmeasurement)−(Body weight of mouse at the time of grouping)]/(Bodyweight of mouse at the time of grouping))×100

TABLE 21 Day 23 Day 46 Group RTV BWC RTV BWC DCR Control 15.66 11.3 1.Paclitaxel 0.69 3.0 5.38 11.6 40% 2. Compound 1 10.47 11.6 3.Comparative Compound 15.23 11.6 1 4. Compound 1/Paclitaxel 0.08 2.2 0.619.4 80% 5. Comparative Compound 0.29 −3.9 3.95 6.7 25% 1/Paclitaxel^(#)^(#)One mouse died out of five mice on day 29

As a result, in comparison with the paclitaxel-only administered group(Group 1), the anti-tumor effect was remarkably potentiated in the groupgiven combination administration of the compound of the presentinvention (Compound 1) and paclitaxel (Group 4) without greatlyincreasing toxicity, which is manifested as, for example, a decrease inbody weight. Also, from the RTV and DCR values on day 46 ofadministration, it was found that a continuous tumor reducing effectcould be expected from the administration of Compound 1. Meanwhile, asapparent from the RTV and DCR values on day 23 and day 46 ofadministration, Comparative Compound 1 did not clearly potentiate theanti-tumor effect when administered in combination with paclitaxel(Group 5), in comparison with the paclitaxel-only administered group(Group 1).

Test Example 5: Evaluation of the Anti-Tumor Effect (2)

By a similar method to that used in Test Example 4, theluciferase-introduced human uterine cervical cancer cell line (Hela-Luc)was subcutaneously transplanted into a nude mouse, and five mice in onegroup were grouped into the single drug administration group andcombination administration group by a randomized stratification method(day 1). In the single drug administration groups, paclitaxel (20 mg/kg)was intravenously administered on day 1. Also, Compound 13 (30 mg/kg) orCompound 22 (100 mg/kg) was orally administered twice a day on day 2 andday 3. In the combination administration groups, paclitaxel (20 mg/kg)was intravenously administered on day 1, and Compound 13 (30 mg/kg) orCompound 22 (60 mg/kg) was orally administered twice a day on day 2 andday 3. The average RTV and BWC values on the 11^(th) day after groupingwere shown in Tables 22 and 23.

TABLE 22 Day 11 Group RTV BWC Control 7.24 7.2 Paclitaxel 2.90 5.4Compound 13 6.54 6.0 Compound 13/Paclitaxel 0.72 4.2

TABLE 23 Day 11 Group RTV BWC Control 5.68 10.9 Paclitaxel 3.05 7.8Compound 22 4.66 6.9 Compound 22/Paclitaxel 1.73 2.8

As a result, in comparison with the paclitaxel-only administered group,the anti-tumor effect was remarkably potentiated in the group givencombination administration of Compound 13 or Compound 22, both of whichare the compounds of the present invention, and paclitaxel, withoutgreatly increasing toxicity, which is manifested as, for example, adecrease in body weight.

Test Example 6: Measurement of the Aurora C Kinase Activity

The inhibitory activity of the compound of the present invention on theaurora C kinase activity was measured in vitro. Specifically, using theOff-chip Mobility Shift Assay, reactions were carried out by thefollowing procedure.

To reaction buffer (20 mM HEPES, 0.01% Triton X-100, 2 mM DTT, pH 7.5),the compound of the present invention, ATP (a final concentration of 25μM), substrates (Kemptide, a final concentration of 1000 nM), magnesium(a final concentration of 5 mM), and purified human aurora C kinase wereadded, followed by mixing. The reactions were then allowed to proceed atroom temperature for one hour. To obtain purified human aurora C kinase,the GST protein was fused to the N-terminus of the full-length aurora Ckinase, and the resulting protein was expressed by baculovirus. TheGST-aurora C fused protein was purified by glutathione sepharosechromatography. Upon completion of the reaction, the reactionterminating solution (QuickScout Screening Assist MSA; CarnaBiosciences, Inc.) was added, and the substrate peptide andphosphorylated peptide in the reaction solution were separated andquantitated by the LabChip 3000 system (Carna Biosciences, Inc.). The invitro method for measuring the inhibitory activity of the compound ofthe present invention on the aurora C kinase activity was performed inaccordance with the method for measuring the inhibitory activity onaurora A demonstrated in Test Example 1-2) above, and defining theconcentration of the compound at which the phosphorylation reaction canbe inhibited by 50% as the IC₅₀ value (nM), the resulting inhibitoryactivity on aurora C kinase was compared with the inhibitory activity onaurora A obtained in the aforementioned Test Example 1-2).

As a result, the inhibitory activity of the compound of the presentinvention on aurora C was 80 to 100 times as weak as the inhibitoryactivity on aurora A, suggesting a significant aurora A-selectiveinhibitory activity of the compound of the present invention.

Test Example 7: Action of Potentiating the Effect ofMicrotubule-Targeting Agents (In Vitro)

Cells of the human-derived stomach cancer cell line OCUM-2M and thehuman-derived uterine cancer cell line HeLa were routinely subculturedin the 10% fetal bovine serum (FBS)-containing Dulbecco's Modified EagleMedium (DMEM) at a cell density of no more than 80%. In order toinitiate a test for the inhibitory activity on cell proliferation, cellswere each suspended in the aforementioned medium and seeded in each wellof a 96-well flat bottom plate (black plate with a transparent bottom)at 2,500 or 3,000 cells per well. The cells were then cultured for oneday at 37° C. in an incubator containing 5% carbon dioxide gas. The nextday, serially diluted solutions of microtubule-targeting agents(docetaxel, cabazitaxel, and epothilone B) were prepared with DMSO (10doses were prepared for each drug at a test concentration ranging from0.03 nM to 1000 nM), and the solutions were diluted with the medium.Subsequently, the resulting solutions were added to each well of thecell culture plate at a final DMSO concentration of 0.1%. Also, in orderto verify the combined effect of the microtubule-targeting agent and thecompound of the present invention, the aforementioned compound wasdiluted with DMSO at a final concentration of 300 nM (a final DMSOconcentration of 0.1%), and then added to each well of the cell cultureplate. As the comparative control group, wells each containing themicrotubule-targeting agent alone or the present compound alone wereseparately prepared and cultured in an incubator containing 5% carbondioxide gas at 37° C. for 72 hours. The cells were counted using theCellTiter-Glo Luminescent Cell Viability Assay kit (the product ofPromega) based on the protocol recommended by Promega. The reagentincluded in the kit was added to each plate, followed by stirring, andthe plates were left to stand at room temperature for 10 minutes. Uponcompletion of the reaction, the luminescence signal was measured using amicroplate reader.

The cell proliferation rate was calculated from the following formula,and the concentration of the test compound at which the cellproliferation was inhibited by 50% (IC₅₀ (μM)) was determined.

Cell proliferation rate (%)=T/C×100

T: Luminescence signal in a well with the addition of the test compoundC: Luminescence signal in a well without the addition of the testcompound

Further, the IC₅₀ value of the microtubule-targeting agent alone (IC₅₀_(_)microtubule-targeting agent) and the IC₅₀ value of themicrotubule-targeting agent in the combination administration of themicrotubule agonist and the compound of the present invention (IC₅₀_(_)combination administration) were determined. The latter IC₅₀ value(IC₅₀ value_combination administration) was calculated from the cellproliferation-inhibitory rate (converted value) in the combinationadministration group by defining the cell proliferation rate of thecompound of the present invention alone as 100%. The degree ofpotentiation of the effect of the microtubule-targeting agent by theaddition of the compound of the present invention was evaluated by thedegree of the value calculated from the following formula.

(Potentiating effect of the combination administration)=(IC₅₀value_combination administration)/(IC₅₀ value_microtubule-targetingagent)

The evaluation was performed according to criteria by which the value ofmore than 1 was evaluated as exhibiting strong potentiating effect,while the value of less than or equal to 1 was evaluated as exhibitingpoor potentiating effect.

As a result, by adding the compound of the present invention tomicrotubule-targeting agents such as docetaxel, cabazitaxel, andepothilone B, a value of larger than 2 was obtained from the aboveformula, indicating that the compound of the present inventionpotentiated the inhibitory effect of these microtubule agonists on cellproliferation.

As shown above, the compound of the present invention selectively andexcellently inhibited aurora A, showing an excellent anti-tumor effectwith favorable oral absorption. Also, the compound of the presentinvention was shown to strongly potentiate the anti-tumor effect ofpaclitaxel by in vivo tests using nude mice. Furthermore, the compoundof the present invention was shown to potentiate also the inhibitoryeffect of microtubule-targeting agents other than paclitaxel on cellproliferation.

1. (canceled)
 2. A method for potentiating an anti-tumor effect of amicrotubule-targeting agent, comprising: administering an effective doseof a piperidine compound of formula (I) or a salt thereof:

wherein R₁ is a carboxyl group, —C(═O)NR₅R₆, or an oxadiazolyl groupoptionally having a C₁-C₆ alkyl group or a trifluoromethyl group as asubstituent; R₂ is a halogen atom or a C₁-C₆ alkoxy group; R₃ is aphenyl group optionally having 1 to 3 same or different group(s)selected from the group consisting of a halogen atom, a C₁-C₆ alkylgroup, a C₁-C₆ alkoxy group, and a trifluoromethyl group as asubstituent; R₄ is a hydrogen atom or a C₁-C₆ alkyl group; R₅ and R₆ arethe same or different and are each independently a hydrogen atom, aC₁-C₆ alkyl group, or a C₃-C₆ cycloalkyl group, or R₅ and R₆ optionallyform a 3 to 6-membered nitrogen-containing saturated heterocyclic grouptogether with a nitrogen atom to which R₅ and R₆ are bound.
 3. Themethod according to claim 2, wherein the anti-tumor effect ispotentiated on cancer which is one of head and neck cancer, esophagealcancer, stomach cancer, duodenal cancer, colon cancer, rectal cancer,liver cancer, gallbladder and bile duct cancer, biliary tract cancer,pancreatic cancer, lung cancer, breast cancer, ovary cancer, uterinecervical cancer, uterine cancer, renal cancer, bladder cancer, prostatecancer, testicular cancer, bone and soft tissue sarcoma, hematologiccancer, multiple myeloma, skin cancer, brain tumor, mesothelioma, andhematologic cancer.
 4. The method according to claim 2, wherein theanti-tumor effect is potentiated on cancer which is one of hematologiccancer, uterine cervical cancer, stomach cancer, breast cancer, prostatecancer, ovary cancer, lung cancer, and colon cancer.
 5. The methodaccording to claim 2, wherein the microtubule-targeting agent is ataxane anticancer agent.
 6. The method according to claim 5, wherein thetaxane anticancer agent comprises at least one selected from the groupconsisting of paclitaxel, docetaxel, and cabazitaxel.
 7. The methodaccording to claim 2, wherein the administering is performed such that amolar ratio of the microtubule-targeting agent and the compound of theformula (I) or a salt thereof is from 0.01 to
 100. 8. The methodaccording to claim 2, wherein the administering is performed such that amolar ratio of the microtubule-targeting agent and the compound of theformula (I) or a salt thereof is from 0.05 to
 50. 9. The methodaccording to claim 2, wherein the administering is performed such that amolar ratio of the microtubule-targeting agent and the compound of theformula (I) or a salt thereof is from 0.1 to
 20. 10. The methodaccording to claim 2, wherein the piperidine compound of formula (I) isone of1-(2,3-dichlorobenzoyl)-4-((5-fluoro-6-(5-methyl-1H-pyrazol-3-ylamino)pyridin-2-yl)methyl)piperidine-4-carboxylicacid,1-(2-fluoro-3-trifluoromethylbenzoyl)-4-((5-fluoro-6-(5-methyl-1H-pyrazol-3-ylamino)pyridin-2-yl)methyl)piperidine-4-carboxylicacid,1-(3-chloro-2-fluorobenzoyl)-4-((5-fluoro-6-(1H-pyrazol-3-ylamino)pyridin-2-yl)methyl)piperidine-4-carboxylicacid,1-(3-chloro-2-fluorobenzoyl)-4-((5-methoxy-6-(5-methyl-1H-pyrazol-3-ylamino)pyridin-2-yl)methyl)piperidine-4-carboxylicacid,1-(3-chloro-2-fluorobenzoyl)-4-((5-chloro-6-(5-methyl-1H-pyrazol-3-ylamino)pyridin-2-yl)methyl)piperidine-4-carboxylicacid,1-(3-chloro-2-fluorobenzoyl)-4-((5-fluoro-6-(5-methyl-1H-pyrazol-3-ylamino)pyridin-2-yl)methyl)piperidine-4-carboxylicacid,1-(3-chloro-2-fluorobenzoyl)-4-((5-fluoro-6-(5-methyl-1H-pyrazol-3-ylamino)pyridin-2-yl)methyl)-N-methylpiperidine-4-carboxyamide,1-(3-chloro-2-fluorobenzoyl)-4-((5-fluoro-6-(5-methyl-1H-pyrazol-3-ylamino)pyridin-2-yl)methyl)-N,N-dimethylpiperidine-4-carboxyamide,azetidin-1-yl(1-(3-chloro-2-fluorobenzoyl)-4-((5-fluoro-6-(5-methyl-1H-pyrazol-3-ylamino)pyridin-2-yl)methyl)piperidin-4-yl)methanone,(1-(3-chloro-2-fluorobenzoyl)-4-((5-fluoro-6-(5-methyl-1H-pyrazol-3-ylamino)pyridin-2-yl)methyl)piperidin-4-yl)(isoxazolidin-2-yl)methanone,(3-chloro-2-fluorophenyl)(4-((5-fluoro-6-(5-methyl-1H-pyrazol-3-ylamino)pyridin-2-yl)methyl)-4-(5-methyl-1,2,4-oxadiazol-3-yl)piperidin-1-yl)methanone,(2,3-dichlorophenyl)(4-((5-fluoro-6-(5-methyl-1H-pyrazol-3-ylamino)pyridin-2-yl)methyl)-4-(5-methyl-1,2,4-oxadiazol-3-yl)piperidin-1-yl)methanone,(3-chloro-2-fluorophenyl)(4-((5-fluoro-6-(5-methyl-1H-pyrazol-3-ylamino)pyridin-2-yl)methyl)-4-(1,2,4-oxadiazol-3-yl)piperidin-1-yl)methanone,(3-chloro-2-fluorophenyl)(4-((5-fluoro-6-(5-methyl-1H-pyrazol-3-ylamino)pyridin-2-yl)methyl)-4-(5-methyl-1,3,4-oxadiazol-3-yl)piperidin-1-yl)methanone,and(3-chloro-2-fluorophenyl)(4-((5-fluoro-6-(5-methyl-1H-pyrazol-3-ylamino)pyridin-2-yl)methyl)-4-(3-methyl-1,2,4-oxadiazol-5-yl)piperidin-1-yl)methanone.11. A method for treating cancer, comprising: administering an effectivedose of a piperidine compound of formula (I) or a salt thereof and aneffective dose of a microtubule-targeting agent:

wherein R₁ is a carboxyl group, —C(═O)NR₅R₆, or an oxadiazolyl groupoptionally having a C₁-C₆ alkyl group or a trifluoromethyl group as asubstituent; R₂ is a halogen atom or a C₁-C₆ alkoxy group; R₃ is aphenyl group optionally having 1 to 3 same or different group(s)selected from the group consisting of a halogen atom, a C₁-C₆ alkylgroup, a C₁-C₆ alkoxy group, and a trifluoromethyl group as asubstituent; R₄ is a hydrogen atom or a C₁-C₆ alkyl group; R₅ and R₆ arethe same or different and are each independently a hydrogen atom, aC₁-C₆ alkyl group, or a C₃-C₆ cycloalkyl group, or R₅ and R₆ optionallyform a 3 to 6-membered nitrogen-containing saturated heterocyclic grouptogether with a nitrogen atom to which R₅ and R₆ are bound.
 12. Themethod according to claim 11, wherein the cancer is one of head and neckcancer, esophageal cancer, stomach cancer, duodenal cancer, coloncancer, rectal cancer, liver cancer, gallbladder and bile duct cancer,biliary tract cancer, pancreatic cancer, lung cancer, breast cancer,ovary cancer, uterine cervical cancer, uterine cancer, renal cancer,bladder cancer, prostate cancer, testicular cancer, bone and soft tissuesarcoma, hematologic cancer, multiple myeloma, skin cancer, brain tumor,mesothelioma, and hematologic cancer.
 13. The method according to claim11, wherein the cancer is one of hematologic cancer, uterine cervicalcancer, stomach cancer, breast cancer, prostate cancer, ovary cancer,lung cancer, and colon cancer.
 14. The method according to claim 11,wherein the microtubule-targeting agent is a taxane anticancer agent.15. The method according to claim 14, wherein the taxane anticanceragent comprises at least one selected from the group consisting ofpaclitaxel, docetaxel, and cabazitaxel.
 16. The method according toclaim 11, wherein the administering is performed such that a molar ratioof the microtubule-targeting agent and the compound of the formula (I)or a salt thereof is from 0.01 to
 100. 17. The method according to claim11, wherein the administering is performed such that a molar ratio ofthe microtubule-targeting agent and the compound of the formula (I) or asalt thereof is from 0.05 to
 50. 18. The method according to claim 11,wherein the administering is performed such that a molar ratio of themicrotubule-targeting agent and the compound of the formula (I) or asalt thereof is from 0.1 to
 20. 19. The method according to claim 11,wherein the piperidine compound of formula (I) is one of1-(2,3-dichlorobenzoyl)-4-((5-fluoro-6-(5-methyl-1H-pyrazol-3-ylamino)pyridin-2-yl)methyl)piperidine-4-carboxylicacid,1-(2-fluoro-3-trifluoromethylbenzoyl)-4-((5-fluoro-6-(5-methyl-1H-pyrazol-3-ylamino)pyridin-2-yl)methyl)piperidine-4-carboxylicacid,1-(3-chloro-2-fluorobenzoyl)-4-((5-fluoro-6-(1H-pyrazol-3-ylamino)pyridin-2-yl)methyl)piperidine-4-carboxylicacid,1-(3-chloro-2-fluorobenzoyl)-4((5-methoxy-6-(5-methyl-1H-pyrazol-3-ylamino)pyridin-2-yl)methyl)piperidine-4-carboxylicacid,1-(3-chloro-2-fluorobenzoyl)-4-((5-chloro-6-(5-methyl-1H-pyrazol-3-ylamino)pyridin-2-yl)methyl)piperidine-4-carboxylicacid,1-(3-chloro-2-fluorobenzoyl)-4-((5-fluoro-6-(5-methyl-1H-pyrazol-3-ylamino)pyridin-2-yl)methyl)piperidine-4-carboxylicacid,1-(3-chloro-2-fluorobenzoyl)-4-((5-fluoro-6-(5-methyl-1H-pyrazol-3-ylamino)pyridin-2-yl)methyl)-N-methylpiperidine-4-carboxyamide,1-(3-chloro-2-fluorobenzoyl)-4-((5-fluoro-6-(5-methyl-1H-pyrazol-3-ylamino)pyridin-2-yl)methyl)-N,N-dimethylpiperidine-4-carboxyamide,azetidin-1-yl(1-(3-chloro-2-fluorobenzoyl)-4-((5-fluoro-6-(5-methyl-1H-pyrazol-3-ylamino)pyridin-2-yl)methyl)piperidin-4-yl)methanone,(1-(3-chloro-2-fluorobenzoyl)-4-((5-fluoro-6-(5-methyl-1H-pyrazol-3-ylamino)pyridin-2-yl)methyl)piperidin-4-yl)(isoxazolidin-2-yl)methanone,(3-chloro-2-fluorophenyl)(4-((5-fluoro-6-(5-methyl-1H-pyrazol-3-ylamino)pyridin-2-yl)methyl)-4-(5-methyl-1,2,4-oxadiazol-3-yl)piperidin-1-yl)methanone,(2,3-dichlorophenyl)(4-((5-fluoro-6-(5-methyl-1H-pyrazol-3-ylamino)pyridin-2-yl)methyl)-4-(5-methyl-1,2,4-oxadiazol-3-yl)piperidin-1-yl)methanone,(3-chloro-2-fluorophenyl)(4-((5-fluoro-6-(5-methyl-1H-pyrazol-3-ylamino)pyridin-2-yl)methyl)-4-(1,2,4-oxadiazol-3-yl)piperidin-1-yl)methanone,(3-chloro-2-fluorophenyl)(4-((5-fluoro-6-(5-methyl-1H-pyrazol-3-ylamino)pyridin-2-yl)methyl)-4-(5-methyl-1,3,4-oxadiazol-3-yl)piperidin-1-yl)methanone,and(3-chloro-2-fluorophenyl)(4-((5-fluoro-6-(5-methyl-1H-pyrazol-3-ylamino)pyridin-2-yl)methyl)-4-(3-methyl-1,2,4-oxadiazol-5-yl)piperidin-1-yl)methanone.